The Interactive Fly

Genes involved in tissue and organ development


What are glia?
Glial migration to the optic lobe is directed by retinal axons
Time-lapse and cell ablation reveal the role of cell interactions in fly glia migration and proliferation
A global in vivo Drosophila RNAi screen identifies a key role of ceramide phosphoethanolamine for glial ensheathment of axons
Gliopodia extend the range of direct glia-neuron communication during the CNS development in Drosophila
Organization and postembryonic development of glial cells in the adult central brain of Drosophila
miR-31 mutants reveal continuous glial homeostasis in the adult Drosophila brain
Analysis of glial distribution in Drosophila adult brains
Multifunctional glial support by Semper cells in the Drosophila retina
Glial glycolysis is essential for neuronal survival in Drosophila
Cyclin-dependent kinase 9 is required for the survival of adult Drosophila melanogaster glia
Antioxidant role for lipid droplets in a stem cell niche of Drosophila
Monitoring cell-cell contacts in vivo in transgenic animals
Mactosylceramide prevents glial cell overgrowth by inhibiting insulin and fibroblast growth factor receptor signaling
TRAP-seq profiling and RNAi-based genetic screens identify conserved glial genes required for adult Drosophila behavior
Tyramine actions on Drosophila flight behavior are affected by a glial dehydrogenase/reductase
The glia-geuron lactate shuttle and elevated ROS promote lipid synthesis in neurons and lipid droplet accumulation in glia via APOE/D
Chaski, a novel Drosophila lactate/pyruvate transporter required in glia cells for survival under nutritional stress
The sulfite oxidase Shopper controls neuronal activity by regulating glutamate homeostasis in Drosophila ensheathing glia
Glial Ca(2+) signaling links endocytosis to K(+) buffering around neuronal somas to regulate excitability
Inhibiting glutamate activity during consolidation suppresses age-related long-term memory impairment in Drosophila
The pleiotropic effects of Innexin genes expressed in Drosophila glia encompass wing chemosensory sensilla
Neuronal lactate levels depend on glia-derived lactate during high brain activity in Drosophila
SIK suppresses neuronal hyperexcitability by regulating the glial capacity to buffer K(+) and water
Accumulation of laminin monomers in Drosophila glia leads to glial endoplasmic reticulum stress and disrupted larval locomotion
A miRNA screen procedure identifies garz as an essential factor in adult glia functions and validates Drosophila as a beneficial 3Rs model to study glial functions and GBF1 biology

Glia and the Blood-Brain Barrier
Organization and function of the blood-brain barrier in Drosophila
The Drosophila blood-brain barrier as interface between neurons and hemolymph
Polyploidization of glia in neural development links tissue growth to blood-brain barrier integrity
Dynamic analysis of the mesenchymal-epithelial transition of blood-brain barrier forming glia in Drosophila
Differential expression of the Drosophila Ntan/Obek controls ploidy in the blood-brain barrier
SIK suppresses neuronal hyperexcitability by regulating the glial capacity to buffer K(+) and water
Accumulation of laminin monomers in Drosophila glia leads to glial endoplasmic reticulum stress and disrupted larval locomotion

Glia, ensheathment, axon pruning, phagocytosis, engulfment and cell death
Cell death triggers olfactory circuit plasticity via glial signaling in Drosophila
Loss of focal adhesions in glia disrupts both glial and photoreceptor axon migration in the Drosophila visual system
Engulfing action of glial cells is required for programmed axon pruning during Drosophila metamorphosis
Involvement of glia in motor neuron retraction during metamorphosis; Mediation by inputs from TGF-beta/BMP signaling and orphan nuclear receptors
DRK/DOS/SOS converge with Crk/Mbc/dCed-12 to activate Rac1 during glial engulfment of axonal debris
Remodeling of peripheral nerve ensheathment during the larval-to-adult transition in Drosophila
Loss- or gain-of-function mutations in ACOX1 cause axonal loss via different Mechanisms

Glial stem cells and the development of glial types
Subtypes of glial cells in the Drosophila embryonic ventral nerve cord as related to lineage and gene expression
Origins of glial cell populations in the insect nervous system
Distinct types of glial cells populate the Drosophila antenna
Multipotent neural stem cells generate glial cells of the central complex through transit amplifying intermediate progenitors in Drosophila brain development
Regulation of Drosophila glial cell proliferation by Merlin-Hippo signaling
Lineage-guided Notch-dependent gliogenesis by Drosophila multi-potent progenitors
Concerted control of gliogenesis by InR/TOR and FGF signalling in the Drosophila post-embryonic brain
Antagonistic feedback loops involving rau and sprouty in the Drosophila eye control neuronal and glial differentiation
Chromatin remodeling during the in vivo glial differentiation in early Drosophila embryos
The secreted neurotrophin Spatzle 3 promotes glial morphogenesis and supports neuronal survival and function
Differing strategies despite shared lineages of motor neurons and glia to achieve robust development of an adult neuropil in Drosophila
Multiple lineages enable robust development of the neuropil-glia architecture in adult Drosophila
A miRNA screen procedure identifies garz as an essential factor in adult glia functions and validates Drosophila as a beneficial 3Rs model to study glial functions and GBF1 biology

Astrocytes engage unique molecular programs to engulf pruned neuronal debris from distinct subsets of neurons
Astrocytes play a key role in Drosophila mushroom body axon pruning
Neuron-glia interactions through the Heartless FGF receptor signaling pathway mediate morphogenesis of Drosophila astrocytes
Astrocytic glutamate transport regulates a Drosophila CNS synapse that lacks astrocyte ensheathment
The astrocyte network in the ventral nerve cord neuropil of the Drosophila third-instar larva

Cortex Glia
Drosophila cortex and neuropile glia influence secondary axon tract growth, pathfinding, and fasciculation in the developing larval brain
Defective cortex glia plasma membrane structure underlies light-induced epilepsy in cpes mutants
Cortex glia clear dead young neurons via Drpr/dCed-6/Shark and Crk/Mbc/dCed-12 signaling pathways in the developing Drosophila optic lobe

Ensheathing Glia
Ensheathing glia function as phagocytes in the adult Drosophila brain
Fibroblast growth factor signaling instructs ensheathing glia wrapping of Drosophila olfactory glomeruli

Midline Glia
Time-lapse imaging reveals stereotypical patterns of Drosophila midline glial migration

Perineural Glia
Identity, origin, and migration of peripheral glial cells in the Drosophila embryo
Perineurial Barrier Glia Physically Respond to Alcohol in an Akap200-Dependent Manner to Promote Tolerance

Wrapping Glia
Wrapping glial morphogenesis and signaling control the timing and pattern of neuronal differentiation in the Drosophila lamina

Glia and the Neuromuscular Junction
Glial wingless/Wnt regulates glutamate receptor clustering and synaptic physiology at the Drosophila neuromuscular junction
Glial response to hypoxia in mutants of NPAS1/3 homolog Trachealess through Wg signaling to modulate synaptic bouton organization

Genes influencing glial differentiation

What are glia?

Glia constitute a support system for neurons. Neurons process and carry information from one neuron to the next, or between neurons and muscles. In their supportive role glia provide neurons with nutrition and growth factors; they maintain neural homeostasis by removing toxic substances, and provide neurons with electrical insulation, assuring proper neuron function. In addition, glia act as substrates for neural migration.

Subtypes of glial cells in the Drosophila embryonic ventral nerve cord as related to lineage and gene expression

In the Drosophila embryonic CNS several subtypes of glial cells develop, which arrange themselves at characteristic positions and presumably fulfil specific functions. The mechanisms leading to the specification and differentiation of glial subtypes are largely unknown. By DiI labelling in glia-specific Gal4 lines the lineages of the lateral glia in the embryonic ventral nerve cord were clarified and each glial cell was linked to a specific stem cell. For the lineage of the longitudinal glioblast, it was shown to consist of 9 cells, which acquire at least four different identities. A large collection of molecular markers (many of them representing transcription factors and potential Gcm target genes) reveals that individual glial cells express specific combinations of markers. However, cluster analysis uncovers similar combinatorial codes for cells within, and significant differences between the categories of surface-associated, cortex-associated, and longitudinal glia. Glial cells derived from the same stem cell may be homogeneous (though not identical; stem cells NB1-1, NB5-6, NB6-4, LGB) or heterogeneous (NB7-4, NB1-3) with regard to gene expression. In addition to providing a powerful tool to analyse the fate of individual glial cells in different genetic backgrounds, each of these marker genes represents a candidate factor involved in glial specification or differentiation. This was demonstrated by the analysis of a castor loss of function mutation, which affects the number and migration of specific glial cells (Beckervordersandforth, 2008).

This report provides a comprehensive description of marker gene and enhancer trap expression in CNS glial cells of late Drosophila embryos. The markers include many transcription factors known to be involved in cell fate specification, as well as a number of still unknown factors. They were chosen for this analysis either because they were known to be expressed in subsets of glial cells or because they were known to be involved in cell fate determination in the nervous system. All together, more than 50 markers were tested, 39 of which showed expression in glial cells and hence were described in detail. Their specific expression patterns, though in many cases not restricted to glia, enable identification of groups of cells, as well as individual cells (Beckervordersandforth, 2008).

The lateral CNS glial cells have been assigned to three categories, according to their spatial distribution and morphology: the surface-, the cortex-, and the neuropile-associated glial cells. Categories were further divided into subgroups, as for example the surface-associated glial cells into subperineural glial cells and channel glia. Several of the molecular markers described in this study exhibit expression patterns, which correspond to the spatial/morphological definition of glial categories or subgroups. For example, moody or svp-lacZ are expressed in all surface-associated glial cells, whereas P101-lacZ is only expressed in the SPG-subgroup and engrailed only in the CG-subgroup. In addition, nearly each individual glial cell expresses a specific combination of markers indicating that they develop unique identities. Yet, nothing is known about how these identities are acquired. Comparing subtype affiliation or lineage ancestry of all glial cells with their respective marker gene expression patterns, it becomes obvious that glial cell specification is a process occurring on the level of individual cells. Cells might have a predisposition for a particular subtype laid down by lineage (e.g. NB5-6 derived cells to become subperineurial glia). In contrast, a temporal cascade within a lineage could determine individual cell identities (as it might be the case for the NB7-4 lineage) (Beckervordersandforth, 2008).

DiI labelling of the lineages of various progenitor cells in combination with cell-specific enhancer trap lines revealed that the composition of glial progeny within the lineages is invariant. Clonally related glia cells often express similar combinations of marker genes. The LGs, a prominent subgroup of the neuropile-associated glia and the only interface glia in the embryonic VNC, have been defined as the progeny of the LGB, which become aligned along the longitudinal connectives. However, there has been confusion about the size and composition of the LGB-lineage. By means of DiI labelling and marker gene expression, the size of this lineage was determined to be 9 cells. Although all cells of the LGB-lineage express a similar set of markers, a few markers are restricted to only parts of the lineage. Based on such markers, as well as positional criteria, the group of LGs was further subdivided. One of the cells, the LP-LG, is located slightly more lateral than the other LGs, and seems to be geared towards the ISN. Since it lies close to, and expresses a similar combination of markers as the M-ISNG, it would be justified to assign this cell to the group of nerve root glia; however, this was not done in order to avoid conflicts with the established nomenclature. Despite of their similarities, these two cells are of different origin: the LP-LG derives from the LGB, and the M-ISNG is generated by NB1-3 (Beckervordersandforth, 2008).

Most of the NBs that arise at corresponding positions and times in thoracic and abdominal segments (called serially homologous NBs) acquire the same fate, i.e. they generate the same lineages expressing corresponding sets of markers. However, some of these serially homologous lineages develop characteristic, tagma-specific differences with regard to cell number and/or cell types. Tagma-specific characteristics of these lineages have been shown to be under the control of Hox genes (Beckervordersandforth, 2008).

Although the total number of CNS glial cells is identical in thoracic and abdominal neuromeres, there are some differences in their origin and distribution of subtypes. This is due to tagma-specific differences among serially homologous lineages of NBs 1-1, 2-2, 5-6, and 6-4, which give rise to CBGs and SPGs. NB6-4A (A, abdominal) generates only two CBGs: MM-CBG and M-CBG, whereas the NB6-4T (T, thoracic) lineage comprises an additional MM-CBG2 and a neuronal sublineage. NB1-1T generates only neurons, whereas NB1-1A produces three SPGs (A-SP-G, B-SPG, and LV-SPG) in addition to neurons. In the thorax, the LV-SPG is presumably generated by NB5-6T, a cell at the position of A-SPG is produced by NB2-2T, and a cell at the B-SPG position is missing. Despite their different origin, the NB1-1A- and NB2-2T-derived SPGs specifically express hkb-lacZ and mirr-lacZ. Furthermore, the NB1-1A- and the NB2-2T-derived A-SPG appear to assume the same identity, as they express the same set of markers (including castor, which is not found in the abdominal B-SPG. Taken together, the differences between thorax and abdomen are restricted to only few glial cells, most of which acquire similar cell fates in thorax and abdomen (as judged by marker gene expression) irrespective of their progenitor (Beckervordersandforth, 2008).

The collection of marker genes and enhancer trap lines presented in this study provides powerful tools for the identification of specific glial cells in different genetic backgrounds. Each of markers also represents a candidate factor involved in glial subtype specification and/or differentiation. Many of these genes encode transcription factors known to be involved in cell fate specification, like fushi tarazu, mirror, and muscle segment homeobox. Other genes encode factors involved in cell signalling, e.g moody and CG11910, or enzymes like CG7433 and CG6218 (Beckervordersandforth, 2008).

moody is expressed in all cells belonging to the surface-associated glia. At the end of embryogenesis, surface-associated glial cells form a thin layer ensheathing the entire CNS, thereby establishing the blood-brain barrier. Moody is a G-protein coupled receptor, which acts in a complex pathway to regulate the cortical actin, thereby stabilizing the extended morphology of the surface-glia. This is necessary for the formation of septate junctions to achieve proper sealing of the nerve cord (Bainton, 2005; Daneman, 2005; Schwabe, 2005). Moody therefore represents a protein, which is essential for establishing and maintaining a specific function of surface glia (Beckervordersandforth, 2008).

Two of the markers analyzed represent metalloproteases: Neprilysin4 (Nep4) and Invadolysin. Invadolysin has been described to play a role in mitotic progression and in migration of germ cells (McHugh, 2004), but as for Nep4, its function in the nervous system is unknown. In vertebrates it has been shown that metalloproteases are involved in various processes in the CNS: they are associated with neurite outgrowth, migration of neurons and myelination of axons. For one matrix-metalloprotease, MMP-12, it has been shown that it is expressed in oligodendrocytes, where it functions in maturation and morphological differentiation of OL lineages (Larsen, 2004; Larsen, 2006). It has been postulated that LGs are analogous to vertebrate oligodendrocytes (Hidalgo, 2000), as both groups of cells enwrap axonal projections in the CNS, although to different degrees (no myelination in Drosophila). The two metalloproteases analysed are exclusively expressed in neuropile- (LGs) and cortex-associated glial cells (CBGs). Thus, both Nep4 and Invadolysin may possibly be involved in the differentiation of LGs. In invadolysin loss of function mutants, the specification of lateral glial cells does not seem to be affected, but the LGs show a very subtle phenotype in their positioning (data not shown). An explanation for the subtle phenotype may be redundant function of both enzymes. Indeed, in vertebrates it has been shown that metalloproteases have many overlapping substrates in vitro, and redundancy and compensation has been shown for matrix-metalloproteases (MMPs) in double mutants. Furthermore, it has been shown for members of the neprilysin family of metalloendopeptidases in Caenorhabditis elegans and Drosophila melanogaster, that many of the enzymatic properties have been conserved during evolution (Beckervordersandforth, 2008).

Making use of the molecular markers, this study characterized the phenotype of a cas loss of function mutation. Cas is a transcription factor, which acts in temporal cell fate specification. Together with Pdm, Cas is involved in the determination of late progeny cells in CNS lineages. In late embryonic stages, cas is specifically expressed in four glial cells per hemisegment, the V-CG and D-CG, the A-SPG and the LV-SPG, as well as in many neurons. The A- and LV-SPG, which are late progeny of the NB1-1A, are not affected in cas mutants, whereas the NB7-4-derived CGs seem mislocalized, with the medial migration of both CGs being impaired in cas mutants. This points to different functions of Cas in distinct NB lineages. As can be deduced from Repo stainings, general aspects of glial differentiation do not seem to be affected in cas mutants. Further analysis will have to clarify whether the role of Cas in NB7-4 derived glial cells is on the level of cell fate determination and/or whether it directly acts on specific aspects of differentiation (migration, motility). It also remains to be shown whether Cas acts cell-autonomously in this process (Beckervordersandforth, 2008).

Developmental biology of glia

Glia origins and diversity mirror that of the neurons they accompany. Longitudinal glial of the ventral nervous system (CNS) are derived from longitudinal gliobasts that have a neuroectodermal origin. Other CNS and brain glia are derived from the same neuroblasts that give rise to neurons. Midline glia have a separate mesectodermal origin, being derived from midline neuroblasts. There are at least two other classes of glia sharing precursors with neurons: those associated with peripheral axons and those associated with sensory organ neurons (Nelson, 1994 and Jones, 1995).

Glial migration to the optic lobe is directed by retinal axons

In the developing Drosophila visual system, glia migrate into stereotyped positions within the photoreceptor axon target fields and provide positional information for photoreceptor axon guidance. Conversely, glial migration depends on photoreceptor axons, as glia precursors stall in their progenitor zones when retinal innervation is eliminated. These results support the view that this requirement for retinal innervation reflects a role of photoreceptor axons in the establishment of an axonal scaffold that guides glial cell migration. Optic lobe cortical axons extend from dorsal and ventral positions toward incoming photoreceptor axons and establish at least four separate pathways that direct glia to proper destinations in the optic lobe neuropiles. Photoreceptor axons induce the outgrowth of these scaffold axons. Most glia do not migrate when the scaffold axons are missing. Moreover, glia follow the aberrant pathways of scaffold axons that project aberrantly, as occurs in the mutant dachsous. The local absence of glia is accompanied by extensive apoptosis of optic lobe cortical neurons. These observations reveal a mechanism for coordinating photoreceptor axon arrival in the brain with the distribution of glia to multiple target destinations, where they are required for axon guidance and neuronal survival (Dearborn, 2004).

The Drosophila optic ganglia are derived from a pair of neuroectodermal proliferative centers known as the inner and outer anlage, located on the ventrolateral surface of each larval brain hemisphere. The onset of Wingless (Wg) expression has been described in a few cells located at the presumptive dorsal and ventral margins of the disc-shaped outer anlagen in young larvae. These two `Wg domains' are positioned as adjacent wedges within the disc-shaped outer anlage. In mid-metamorphosis, the disc opens at a fissure located between the two Wg domains; the disc unfurls linearly, such that the Wg domains move to the future dorsal and ventral poles of the ganglion. Interestingly, the Wg domains are roughly coincident with sites revealed by clonal analyses to be the origins of glia that migrate into the lamina neuropile. This relationship was investigated in greater detail (Dearborn, 2004).

A marker of wingless expression, the lacZ reporter construct 17en40 (or wg-lacZ) was used to examine wg expression with respect to glial cell development and movement. In cells expressing wg-lacZ, cytoplasmic ß-galactosidase fills cellular processes and permits the visualization of their detailed morphology. The paths of glial cell migration are observed to coincide with a stereotyped pattern of cytoplasmic extensions emanating from wg-lacZ expressing cells. The extensions follow stereotyped routes and terminate at specific destinations in the lamina, medulla and lobula. Glia form chains along these extensions and accumulate in neuropile destinations beyond the extension's termini. Marker analysis indicates that the glia undergo differentiation as they progress along these paths. Lamina glia express the early glial differentiation factor glial cells missing (gcm) at their points of entry onto the extensions. Further along the path toward the neuropile, glia commence expression of the homeodomain protein Repo, a marker downstream of Gcm expression. Thus, in the case of lamina marginal glia, differentiation can be assessed relative to glial progression along the migratory pathway (Dearborn, 2004).

The wg-lacZ positive extensions are evidently axons. Their cell membranes label by anti-horseradish peroxidase antibody, a neuronal marker. Their cell bodies label positive for Elav, also a neuronal marker. The axons extending toward the same neuropile destination bundle together in a fascicle, as indicated by labeling of individual axons in mosaic animals. Four wg-lacZ labeled fascicles were resolved emerging from each of the dorsal and ventral Wg domains. One fascicle from each domain extends to the border of the lamina field, terminating at a position adjacent to layers of glia known as the lamina epithelial (Ep) and marginal (Ma) glia (established nomenclature is found at Flybrain). These layers of glia lie, respectively, above and below the layer of the axon termini of the R1-R6 photoreceptors. Glia can be observed migrating in a chain along the fascicles. Owing to the absence of specific markers, epithelial and marginal glia could not be distinguished prior to their separation into distinct layers. It seems likely, however, that both glial types migrate on the same pathway. One fascicle from each Wg domain extends to the cortex, neuropile boundary of the medulla, and is associated with the chain-like migration of medulla neuropile (MNG) glia. One fascicle from each domain extends to the boundary between the medulla and lobula, and corresponds to a pathway for migration of inner chiasm (Xi) glia, which demarcate the border between medulla and lobula neuropiles. The final pair of fascicles extends into the lobula neuropile and forms a pathway associated with lobula neuropile glia (LoG). These four sets of putative migratory guides are referred to as 'scaffold axons'. Notably, the migration of these four glia types depends on retinal innervation of the optic lobe, a requirement that is explored in this study (Dearborn, 2004).

Earlier stage specimens were examined in order to determine the temporal relationship between glial migration and the outgrowth of scaffold axons. During the first two larval stages, most neuroectodermal cells of the outer anlagen express the cell adhesion protein Fasciclin 2 (Fas2), with the exception of those in the two Wingless domains; Wingless-expressing cells are Fas2 negative. As development proceeds to the third instar stage, the neuroectodermal populations mostly convert to blast cells that produce the neurons and glia of the lamina and medulla. The differentiation of wg-lacZ positive scaffold neurons and the extension of their axons follow this general temporal scheme, a small population of glia precedes the arrival of the first photoreceptor axons in the target field of the retinal axon. A small population of centrally located glia have been described that precede the arrival of photoreceptor axons in the lamina. At these early time points, when the Wg domains consist of fewer than a hundred cells; scaffold axon fascicles are not detected. Photoreceptor axons subsequently arrive in the temporal order that follows the posterior to anterior pattern of eye development. The elaboration of wg-lacZ positive scaffold axons is first detected as the first photoreceptor axons arrive in the target field, when only the first one or two columns of ommatidia have initiated differentiation in the eye disc. As retinal innervation continues, the number of glia in the neuropile regions increases steadily. Thus, Wingless-positive cells, though present long before the arrival of photoreceptor axons in the brain, appear to first extend axons when retinal axons begin to arrive in the brain (Dearborn, 2004).

The wg-lacZ labeled-axons also were examined in pupal stage animals, where mature axon projections into the optic lobe neuropiles can be resolved. wg-lacZ positive axons can still be detected extending from cell bodies located at dorsal and ventral cortical positions. The axons project, respectively, to dorsal and ventral targets in the medulla and lobula neuropiles. It is evident that the wg-lacZ-positive neurons include intrinsic neurons of the proximal medulla (Pm neurons), which extend arbors tangentially within small regions of the proximal medulla. Projections into a specific tangential layer of the lobula were also observed. Projections into the lamina neuropile were not observed; at the third instar stage the extensions terminate at the border of the developing lamina neuropile. These axons thus may not have become part of lamina circuitry, or alternatively have ceased wg-lacZ expression at the pupal stages examined (Dearborn, 2004).

Attempts were made to determine the location of progenitors that give rise to the distinct types of migratory glia and the neurons that form their migratory pathways. The Wingless expressing cells of the dorsal and ventral domains are located in areas of complex gene expression controlled by Wingless (Wg) signaling activity. Adjacent to the Wg domains are non-overlapping cell populations that express the TGF-ß family member Decapentaplegic (Dpp). Both the Wg- and Dpp-positive cell populations express the transcription factor Optomotor Blind (Omb). Dachsous (Ds), a Cadherin family member, is expressed in a graded fashion with respect to the Wg domains. These three genes, though expressed in different patterns, are under the control of Wg activity (Dearborn, 2004 and references therein).

By performing clonal analysis with tissue labeled to provide positional landmarks, it was possible to localize glial progenitors and scaffold neurons to distinct sites of origin. The FLP/FRT system was used to generate somatic clones that were positively marked by membrane-bound GFP (UAS-CD8::GFP). Rare recombination events were induced such that most specimens harbored only one or a few labeled cell clones in the developing optic ganglia, which were examined in late third instar larvae. Clones including lamina marginal and epithelial glia were found to label progenitors located at the dorsal and ventral margins of the outer anlagen. Clones that included lamina epithelial glia, lamina marginal glia, medulla neuropile glia, inner chiasm glia or lobula neuropile glia were all found within the domain of cells that express Wg, Omb and Ds (Domain I). A majority of these clones contained both labeled scaffold neurons and glia (16/26); clones with only neurons or glia were less frequent. In the majority of specimens in which glia were labeled, the clone extended into multiple domains and included Domain I. With rare exception, these larger clones also contained neurons. Thus, at the time that somatic recombination was induced (mid-second instar, starting 75 hours after egg laying), most progenitor cells retained the potential to produce both neurons and glia. When the specimens were analyzed with respect to the glial types that were labeled, an interesting pattern emerged with regard to the position of labeled progenitor cells in the Domain I region. Labeled progenitors for each of the glial cell types appeared in distinct domains on the proximal-distal axis. For example, the lamina epithelial and marginal glial progenitors were found in a more lateral position than medulla neuropile glial progenitors. Inner chiasm glial progenitors were observed in an even more proximal location. Hence, the progenitor domains for distinct glial types appear to be organized into a proximal distal stack within Domain I (Dearborn, 2004).

Labeled scaffold neurons were most often included in the glial clones, and were found in close proximity to glial progenitors that used the axons as migratory guides. In a minority of cases, small clones were recovered which included only scaffold neurons. These clones were contained within Domain I (Wg, Omb, Ds expression) in all but two of nineteen cases. These data do not resolve when the glial and neuronal lineages diverge. However, they do permit the conclusion that glia and the neurons that appear to establish their migratory pathways are generated in close proximity and with a lineage relationship (Dearborn, 2004).

The migration of glia from the prospective dorsal and ventral margins of the developing optic lobe depends on the arrival of photoreceptor axons in the target field. When photoreceptor axons are absent, as occurs in mutants that eliminate ommatidial development (sine oculis, eyes absent and eyeless) most glia remain stalled in their progenitor domains. When photoreceptor axons innervate only part of the lamina field, glia migrate to the region that receives retinal innervation. It has thus been supposed that photoreceptor axons attract glia into the lamina target field. It was thus thought it might be informative to examine the glial migratory scaffold under conditions where glial migration did not occur (Dearborn, 2004).

To this end, the axon scaffold was examined in sine oculis1 (so) and EyelessD (ey) animals. These mutants display variable penetrance, such that photoreceptor neurons can be completely absent, or develop in variably sized clusters in a particular region of the developing retina. A lack of retinal innervation has compound effects on optic lobe development. Lamina neurons fail to develop because of the absence of axon-borne signals. The medulla is greatly reduced in cell number by extensive apoptosis. Employing mosaic analysis, Fischbach and Technau (1984) showed that so1 acts in the eye to bring about these effects on the brain (Dearborn, 2004).

In the current analysis, the scaffold axons were labeled by the expression of wg-lacZ. When photoreceptor axons were absent, the scaffold axons were likewise missing. However, wg-lacZ positive cells seemed to be present in normal number in the Wg domains, and expressed the neuronal HRP antigens. In prior work, a number of markers expressed in the vicinity of the Wg domains were expressed normally in the absence of retinal innervation (e.g., Dpp and Omb). Therefore, it is thought unlikely that retinal innervation is required for the differentiation of these optic lobe neurons. When the scaffold axons were absent in so1 and eyD animals, glia accumulated at the edges of the Wg domains near the point where they would have joined axon fascicles on paths toward neuropile destinations. Most so1 and eyD animals develop part of an eye, such that the corresponding optic lobe receives partial innervation. In these cases, photoreceptor axons project to appropriate retinotopic locations despite the absence of the usual array of neighboring axons. In such specimens, scaffold axons were found only in parts of the brain that received retinal innervation and not in regions that completely lacked innervation. A correlation between retinal innervation, scaffold axon extension and glial migration was found in each of 32 animals with partial eye development restricted to either dorsal or ventral regions. These observations thus argue strongly that scaffold axon outgrowth and glial migration depend on retinal innervation (Dearborn, 2004).

Retinal innervation might elicit scaffold axon outgrowth, thereby establishing a necessary pathway for glial migration. Conversely, glial migration, elicited by retinal innervation directly, might establish a necessary pathway for scaffold axon outgrowth. Two approaches were undertaken in an attempt to resolve this issue. In the first, scaffold axons were eliminated by neuronal expression of activated Ras1 (Ras1N17) in order to determine whether glial migration would occur in their absence. In the second, mutant animals with misdirected scaffold axons were examined in order to determine whether migratory glia follow the aberrant axon projections. Both approaches indicated that scaffold axons are necessary as glial migratory guides (Dearborn, 2004).

Prior work has revealed extensive apoptosis in the optic lobes of 'eyeless' mutants of Drosophila. In the mutant sine oculis (so), mosaic analysis has revealed that extensive cell death in the optic lobe is due to the lack of so function in the retina and not the brain. These and additional observations have led to the conclusion that the optic lobe phenotype of sine oculis is due to lack of retinal innervation. The 'trophic' function of photoreceptor axons could be direct, via provision of a survival factor, or indirect, e.g., by eliciting the migration of glia that provide a survival factor (Dearborn, 2004).

To address the issue, wild-type and so1 animals were examined for the onset of apoptosis by their expression of activated caspase, which can be monitored in Drosophila with an antibody against the activated human caspase 3 protein. In third instar stage wild-type animals, few cortical cells are labeled by the anti-Caspase labeling. By contrast, so1 third instar larval optic lobes display an increased number of caspase 3-positive cells throughout the medulla cortex. Putative apoptotic cells were concentrated in regions where glia were particularly few or absent. Caspase 3-positive cells were also particularly prevalent in cortical areas immediately adjacent to the neuropile. Areas particularly deficient in glia also displayed an irregular neuropile structure. These observations were subjected to a quantitative analysis. Areas of 50 µm2 in 1.3 µm confocal optical sections of the medulla cortex were counted for the number of caspase 3-positive cells. In wild-type animals, an average of 0.6 activated caspase-positive cells were found per 50 µm2 area. No regional differences in the density of apoptotic cells were observed in wild-type animals. so1 specimens displayed an average of 5.5 apoptotic cells in 50 µm2 areas adjacent to medulla neuropile regions that lacked medulla neuropile glia. Elevated apoptosis, an average of 6.5 activated caspase-positive cells, was also observed in distal cortical cell populations whose axons would normally innervate glia-starved regions of neuropile. By contrast, cell populations adjacent to glia-rich regions of medulla in the same so1 animals showed only 2.4 apoptotic cells per 50 µm2, while in the corresponding distal cortical cell populations that innervate these glia-rich regions, only 2.5 apoptotic cells per 50 µm2 were found. Thus, although so1 animals displayed an overall increase in caspase-positive cells, the frequency was significantly greater in cell populations that innervated glia-starved regions of neuropile. The results of this analysis are statistically significant: for medulla neuropile proximal regions within so1 animals, a paired t-test analysis yielded a P-value of 1.3e-06 (comparing glia-poor and glia-rich regions), while the same statistical analysis comparing cortical regions yielded a P value of 4.8e-07. These observations suggest that both local and long-range trophic cues are provided by glia to cortical neurons. Additional studies show that the absence of glia, rather than photoreceptor axons, appears the more likely cause of extensive apoptosis and neural cell loss observed in eyeless strains of Drosophila (Dearborn, 2004).

It is concluded that Drosophila optic lobe glia use axon fascicles as migratory guides and that the extension of these axon fascicles is induced by the ingrowth of photoreceptor axons from the developing retina. The migratory scaffold axons emerge from optic lobe regions that are in close proximity to sites where glial cells originate; both arise in the dorsal and ventral domains where cells express the morphogen Wingless. When the scaffold axons were eliminated by the autonomous expression of an activated Ras transgene, glia failed to migrate and stalled at the borders of their progenitor sites. Extensive cortical cell apoptosis ensued. When the scaffold axons projected aberrantly (in animals mutant for the cadherin Dachsous), glia followed the aberrant routes to incorrect destinations. The longstanding observation that glial migration does not occur in eyeless mutant Drosophila might thus be explained by an indirect mechanism in which innervation controls the establishment of an axon scaffold necessary to direct glial migration (Dearborn, 2004).

The migratory scaffold axons were identified by their cytoplasmic expression of ß-galactosidase from lacZ under the control of a wingless promoter. The neurons are thus residents of the Wg domains, a point additionally supported by labeling small numbers of neurons that projected their axons toward glial destinations. In total, four different wg-lacZ delineated pathways were identified. These appear to account for all the pathways taken by optic lobe glia that have been identified as migratory by clonal studies. Separate pathways were identified for medulla neuropile glia, lobula neuropile and inner chiasm glia. A single scaffold axon pathway was observed leading to the marginal and epithelial glial layers of the lamina, suggesting that both of these glial types follow the same pathway. Perhaps these glia become separated only on the interposition of photoreceptor R1-R6 growth cones as they arrive in the lamina. Whether the epithelial and marginal glia arise from distinct precursors that migrate on the same pathway is unclear. In all cases, glia were observed to form migratory 'chains' along axonal extensions, resembling a similar organization of migratory glia on retinal axons en route from the optic stalk to the eye field and from midline progenitor sites to destinations in the PNS. In pupal stage animals, the wg-lacZ labeled neurons were observed in dorsal and ventral cortical locations, sending projections into neuropile targets consistent with the patterns of glial migration. However, no axons from wg-lacZ positive neurons were observed extending into the lamina neuropile at this stage (Dearborn, 2004).

The optic lobe regions surrounding the Wg domains display complex patterns of gene expression, mainly because of the signaling activity of Wingless. Clonal analysis indicates that all five migratory glial cell types examined arise from these domains. Interestingly, the sites from which particular glia arise are stacked on the proximal distal axis in a manner that correlates with target destinations in the developing ganglia. Thus, for example, somatic clones that label the medulla neuropile glia are located at a position that corresponds to the medial/distal position of MNG glia relative to the lamina and lobula glia. Furthermore, on the basis of their expression of wg-lacZ, as well as clonal analysis, the neurons that extend scaffold axons arise in close proximity to the sites of glial origin. Indeed, as somatic clones induced in mid-second instar larval animals often labeled both scaffold neurons and migratory glia, the glia and neurons must share common progenitors. It is curious that all of these distinct cell types express wingless. These is no evidence that axonally transported Wg functions in optic lobe development, as it does in the development of the neuromuscular junction. Partial elimination of wg+ activity (by the use of a conditional wgts allele) did not result in a specific defect in glial migration in the optic lobe but other possible functions of Wg were not addressed by this analysis (Dearborn, 2004).

These observations suggest a developmental mechanism for the control of glial cell migration that depends on the establishment of an axon scaffold for guidance of migrating glia. In normal development, a small number of glia migrate into the target field of the photoreceptor axon prior to the arrival of the first photoreceptor axons. These glia, which migrate independently of retinal innervation, may serve a necessary early role in photoreceptor axon guidance. They may be targets for the first retinal axons to arrive in the optic lobe, and provide the first signals that differentiate the outgrowth termination points of the R1-R6 and R7/8 axons. It is proposed that the first photoreceptor axons to arrive in the optic lobe elicit outgrowth of the scaffold axons from neurons at the dorsal and ventral margins. Subsequent migration of glia from the dorsal and ventral margin progenitors is then both permitted and directed along the specific pathways of the scaffold. After the erection of the migratory scaffold, glial migration may be independent of continued photoreceptor axon ingrowth. On this point, it is noted that glia migrate into the lamina in approximately normal numbers in hh1 animals, in which ommatidial development ceases after 11-13 columns form at the posterior of the developing retina. Therefore, glial migration may not depend on continued arrival of new retinal axons in the lamina primordium. Nonetheless, this observation cannot rule out the alternative interpretation that retinal axons emit a continuous attractive signal for glial migration that functions in adjunct with the migratory axon scaffold (Dearborn, 2004).

How do photoreceptor axons control the outgrowth of axons from the Wg domains? This does not appear to be a consequence of an affect of retinal innervation on neuronal development in the Wg domains, which appear normal in size and organization in eyeless mutant Drosophila strains. Hedgehog, which is brought into the brain by retinal axons, is not required for the expression of either Wg or Dpp at the dorsal and ventral margins of the optic lobe. It seems that the induction of scaffold axon outgrowth by the photoreceptor axons may be direct, since the outgrowth occurs in the hedgehog mutant, hh1, in which the first steps of lamina neuronal development fail to occur. Thus, one might suppose that retinal axons emit a chemoattractant for scaffold axon outgrowth, either synthesized in the retina or acquired from environmental sources and redistributed by retinal axons (Dearborn, 2004).

The system for glial migration guidance permits diversified cell types to originate from a common site, and yet target specific locations in complex neuropiles. One could imagine that as more complex and diversified neuropiles evolved, relatively simple changes in the developmental pathway of glial progenitors and the projections of scaffold axons would deliver glial support to new structures. This system also has the feature of functioning as a developmental timing 'checkpoint' that fine-tunes the general hormonal coordination of imaginal development. Thus, upon their initial arrival in the brain, retinal axons provide a fine level of local cellular control over the movement of glia, preparing the target field for the next steps of optic lobe development (Dearborn, 2004).

Time-lapse and cell ablation reveal the role of cell interactions in fly glia migration and proliferation

Migration and proliferation have been mostly explored in culture systems or fixed preparations. A simple genetic model, the chains of glia moving along fly wing nerves, is presented to follow such dynamic processes by time-lapse in the whole animal. Glia undergo extensive cytoskeleton and mitotic apparatus rearrangements during division and migration. Single cell labelling identifies different glia: pioneers with high filopodial, exploratory, activity and, less active followers. In combination with time-lapse, altering this cellular environment by genetic means or cell ablation has allowed the role of specific cell-cell interactions to be defined -- (1) neuron-glia interactions are not necessary for glia motility but do affect the direction of migration; (2) repulsive interactions between glia control the extent of movement; (3) autonomous cues control proliferation (Aigouy, 2004).

The fly wing contains two purely sensory nerves that navigate proximally along the so-called L1 and L3 veins, and are lined by glial cells. L3 vein contains three gliogenic sensory organs, L3.3, L3.1 and L3.v, from distal to proximal. Each sensory organ precursor (SOP) divides asymmetrically several times and produces a glial precursor (GP), which repeatedly divides and is therefore called a first order glial precursor (GPI). The GPI, initially located adjacent to the neuronal soma, subsequently moves along the underlying axon. The final number of glial cells per lineage varies stochastically from four to eight (average=six). The whole glial lineage, from GPIs to post-mitotic glial cells, expresses the Repo glial marker (Aigouy, 2004).

On L3, proliferation starts several hours after GPI birth, at around 17 hours after puparium formation (APF), and by 20-22 hours APF all GPIs have divided once. The second round of division takes place by 24 hours APF, and the third by 30 hours APF, with little glial proliferation being observed afterwards. These data are in agreement with the GPI dividing symmetrically twice, to produce two second order and four third order precursors (GPII and GPIII, respectively. The presence of up to eight glial cells suggests that GPIIIs may or may not divide, and/or that cell death takes place throughout the lineage (Aigouy, 2004).

The observation that glia migrate and proliferate to variable extents suggests a role for extrinsic signals and makes it difficult to follow the development of glial lineages in fixed tissues. Therefore an in vivo glial marker was devised by crossing the repo-Gal4 driver with a UAS-GFP reporter (repo-GFP) (Aigouy, 2004).

When L1 glia are compared with fly border cells, functioning during oogenesis, and tracheal cells, differences and similarities become apparent. Border cells rely on an asymmetric structure, the actin based-LCE (long cellular extension), which triggers movement through a grapple and pull process. By contrast, wing pioneer glial cells send out numerous filopodia that dynamically assay the environment in all directions; this is also seen in tracheal cells. This may reflect the fact that border cells form a cluster and move simultaneously, whereas, in the chain of glia, distal cells do not contact pioneer cells and follow the movement of adjacent, more proximal, cells. Based on time-lapse and ablation data, the following model is proposed. Prior to migration, pioneer cells are free to explore the proximal axonal substrate whereas follower cells are submitted to bilateral repulsive glia-glia contacts. As pioneer cells move proximally, they free space at more distal positions. Follower cells rapidly occupy such space, thus freeing even more distal regions. This domino-type of migration proceeds until homogenous axon enwrapping is reached (Aigouy, 2004).

Most exploratory activity is spatially and temporally restricted as it specifically characterises cells that move on naked axons. Whether the presence of pioneer cells at the front of migration is lineage dependent or reliant on extracellular signals remains to be determined. In the future, performing ablations throughout the L1 will help to address the issue of plasticity. The presence of LCE versus pioneer filopodia may reflect different modalities of directional migration. It is known that border cells respond to chemoattractants, whereas glial directional migration may be driven by underlying axons. Interestingly, L3 migrating and proliferating cells all show filopodia, suggesting yet a different mode of migration compared with that of cell chains (L1 glia) and clusters (border cells). Finally, glia, but not tracheal or border cells, divide as they move, suggesting that the formation of a continuous chain along the axon bundle requires both migration and proliferation. In the future, it will be crucial to determine how the two events are coordinated (Aigouy, 2004).

The different features shown by border cells, trachea and glia suggest that cell specification controls motility strategies. The role of cell specification cues is further demonstrated by the fact that, even within glial cells, different lineages display distinct features. While wing GPs divide several times, glia arising from dorsal bipolar dendritic embryonic lineages and microchaete glia do not divide, the latter dying soon after birth. Understanding the molecular pathways specifying migratory and proliferative profiles represents one of the future challenges for developmental biologists (Aigouy, 2004).

Like oligodendrocyte precursors in the rat optic nerve that are controlled by an internal clock, GPIs divide a limited number of times. Different sets of data speak in favour of an internal clock that limits the absolute number of divisions to three. (1) The average number of glia derived by different sensory organs is constant, irrespective of the number of underlying axons (L3.3 and L3.1 glia line one and four axons, respectively). Thus, the number of axons does not control proliferation. (2) Time-lapse data show that divisions within and between lineages are rather synchronous. (3) Mitotic clones lacking the Gcm (Glial cell missing) glial promoting factor in one gliogenic sensory organ result in fewer wing glia, indicating that the remaining cells do not compensate for the missing ones. (4) Ablation data demonstrate a lack of compensatory divisions within and among glial lineages. Whether vertebrate and invertebrate clocks rely on the same signals will be a matter of future studies. In addition to the internal clock, extracellular signals may be at work and may control the fine-tuning of proliferation (four to eight cells per lineage). A role of cell-cell interactions in the control of glial proliferation has also been observed in the fly embryo (Aigouy, 2004).

One of the most peculiar features of glia is that they tend to form a chain of cells. This might suggest that glial cells display affinity for axons as well as for other glial cells, the equilibrium between these affinities dictating the extent of migration and triggering the formation of a continuous glial sheath. However, even in the absence of axons (Notchts1 data), glial cells form a continuous chain, rather than staying as a cluster or moving apart from one another. Thus, axons do not trigger glial cell alignment, clearly showing that glia are endowed with an intrinsic migratory potential. The chain of glia present in Notchts1 wings is unbranched, as if the surrounding vein were providing a physical channel or instructive cues for migration. Although a participation of veins in axonal navigation and/or glia migration cannot be excluded, veinless wings still contain properly organised axons and glia. Moreover, ectopic axons present in the intervein space of hairy-wing wings carry properly lined glial cells, thus indicating that veins are not instructive for glial migration (Aigouy, 2004).

One way to reconcile all data is that different types of interactions take place. In one case, glia tend to fully occupy and enwrap naked axons, probably in response to neuronal signals. In contrast, counteracting interactions are at work between glia. Thus, while affinity between glial cells induces them to stay together, repulsive contacts prevent them from forming a cluster and trigger the formation of a chain. The observation that the cytoplasmic processes of adjacent glia largely overlap is in agreement with this hypothesis, and leads to the proposition that glial cells tend to reduce the extent of contact by sliding over each other. The equilibrium between all these forces allows the formation of a continuous chain and controls the extent of movement, compatibly with the substrate available for migration (Aigouy, 2004).

Glial cells move in a stereotyped direction and, as shown by the ablation data, do not require the presence of guide-post glia to find their way. Instead, neuron-glia interactions affect directional migration. Indeed, both in fly wings and in the zebrafish lateral line, misrouted axons result in redirected glia. Furthermore, in both systems, glial arrest is observed upon axonal arrest. The fact that glia use axons as a substrate suggests an unpredicted axonal feature. Indeed, although the polarised nature of the axon is well characterised with respect to microtubule growth, how does the axon convey directional information to the enwrapping glia? The data clearly show that glial migration relies on complex and dynamic glia-glia and neuron-glia interactions. Establishing time-lapse protocols that simultaneously monitor neurons and glia, or aiming at simultaneously identifying the whole glial population and a subset of glia, will be crucial to gaining a better understanding of the precise nature and role of such homo- and hetero-typic interactions (Aigouy, 2004).

Most migratory cells undergo an epithelial to mesenchymal transition that implies changes in cell polarity. Similarly, glial cells originate through the apico-basal division of the IIb precursor. The newly formed GPIs wait almost ten hours before dividing, whereas GPIIs divide and migrate rapidly soon after birth. By the end of this latency period, the GPI acquires a very polarised shape, and divides along the proximodistal axis. Altogether, these results indicate that a change in cell polarity occurs in the GPI, the cell that starts migrating. Thus, latency probably serves to build up the GPI competence for proliferation and migration (Aigouy, 2004).

The fact that glial cells divide and migrate along the same axis suggests that the signalling pathways controlling cell polarity, division and motility are coordinated. The analysis of mutations affecting these processes will be fundamental for understanding the molecular bases of their integration (Aigouy, 2004).

Time-lapse imaging reveals stereotypical patterns of Drosophila midline glial migration

The Drosophila CNS midline glia (MG) are multifunctional cells that ensheath and provide trophic support to commissural axons, and direct embryonic development by employing a variety of signaling molecules. These glia consist of two functionally distinct populations: the anterior MG (AMG) and posterior MG (PMG). Only the AMG ensheath axon commissures, whereas the function of the non-ensheathing PMG is unknown. The Drosophila MG have proven to be an excellent system for studying glial proliferation, cell fate, apoptosis, and axon-glial interactions. However, insight into how AMG migrate and acquire their specific positions within the axon-glial scaffold has been lacking. This study used time-lapse imaging, single-cell analysis, and embryo staining to comprehensively describe the proliferation, migration, and apoptosis of the Drosophila MG. Three groups of MG were identified that differed in the trajectories of their initial inward migration: AMG that migrate inward and to the anterior before undergoing apoptosis, AMG that migrate inward and to the posterior to ensheath commissural axons, and PMG that migrate inward and to the anterior to contact the commissural axons before undergoing apoptosis. In a second phase of their migration, the surviving AMG stereotypically migrated posteriorly to specific positions surrounding the commissures, and their final position was correlated with their location prior to migration. Most noteworthy are AMG that migrated between the commissures from a ventral to a dorsal position. Single-cell analysis indicated that individual AMG possessed wide-ranging and elaborate membrane extensions that partially ensheathed both commissures. These results provide a strong foundation for future genetic experiments to identify mutants affecting MG development, particularly in guidance cues that may direct migration. Drosophila MG are homologous in structure and function to the glial-like cells that populate the vertebrate CNS floorplate, and study of Drosophila MG will provide useful insights into floorplate development and function (Wheeler, 2012).

While cellular analysis of Drosophila MG development has been studied for greater than 20 years, novel insights were gained in this study through the use of multiple advanced imaging approaches. The most important was the use of time-lapse imaging. This allowed tracking of each MG cell between stages 12 and 17, and revealed their stereotyped migration. Development at earlier times (stages 11 to 12) was assessed by analysis of sim-Gal4 UAS-tau-GFP embryos stained for MG markers. Finally, the visualization of individual MG using argos-G1.1-Gal4 UAS-tau-GFP embryos allowed the extensive cytoplasm/membrane extensions of each AMG to be determined. In addition, the analyses of MG in sagittal view allow for a simplified and more accurate reconstruction of MG migration compared to traditional ventral views. Combined, these approaches resulted in a comprehensive cellular view of MG development that will be the basis for future genetic analyses (Wheeler, 2012).

Using multiple cell markers and confocal analysis, it was possible to accurately count the number of AMG and PMG throughout development. Overall, the numbers agree well with. There are ~8 sim+ mesectodermal cells/segment at embryonic stage 7. During stage 8, these cells synchronously divide during the mitotic cycle 14 division to give rise to ~16 cells/segment. It is hypothesized that these 16 cells are initially committed to become neural precursors, and subsequent Notch signaling acts via lateral inhibition to partition these cells into a population of neural precursors and MG. This process results in 8.8 MG/segment (5.4 AMG and 3.4 PMG) at stage 11. The number of AMG and PMG remains constant through stages 12/5 and 12/3 but between stages 12/3 and 12/0 there is an increase of 2.1 AMG/segment to 7.5, suggesting two divisions on average from existing AMG. Consistent with this view is the presence of anti-phospho-histone-H3 (PH3) positive tau-dense AMG at stage 12/0. However, it is worth noting that AMG number (and presumably divisions) can vary significantly between segments. Thus, mechanistically, AMG can be generated in two ways. First, lateral inhibition mediated by Notch signaling leads to the formation of 5.4 AMG/segment during stages 10 and 11; a recruitment step that likely occurs without cell division. Second, divisions of existing AMG occur at stage 12/0 to generate a total of 7.5 AMG/segment (Wheeler, 2012).

Both ecdysone and vein (vn) signaling may play roles in AMG cell division. Steroids are known to influence glial proliferation and apoptosis in vertebrates. Genetic and in vitro studies also indicate that high levels of ecdysteroids can inhibit proliferation and promote apoptosis of Drosophila embryonic and postembryonic MG. These results suggest that AMG have a tendency to divide, but high levels of ecdysone inhibit division. In the embryo, there is a prominent pulse of ecdysone that peaks at 8 h of development (stage 12) and gradually decreases until hatching. Thus, the presence of high levels of ecdysone may prevent AMG proliferation during late embryogenesis, but may not increase quickly enough to prevent AMG from undergoing division during stage 12. Consistent with this view, mutants of genes involved in ecdysone production (e.g. disembodied, shadow, spook) result in additional embryonic MG. While the presence of ecdysone may inhibit MG proliferation, vn signaling may be required for MG divisions. The vn gene encodes a secreted ligand that binds to Egfr and is a homolog of vertebrate neuregulin, which promotes glial division. vn was previously hypothesized to control AMG division at stage 12, since vn mutant embryos showed a reduction of 2.6 AMG/segment. This is similar to the average increase in AMG due to cell division (2.1 AMG/segment). If vn is required for AMG division throughout development, then division should be absent (regardless of ecdysone levels) when vn expression is absent. This is indeed the case as vn is expressed in the midline and surrounding cells from stages 10 to 12, but is restricted to only a small subset of lateral CNS neuroblasts and neurons after AMG divisions. Taken together, this suggests that AMG proliferation may depend on a balance between positive (vn) and negative (ecdysone) signaling (Wheeler, 2012).

AMG number peaks at stage 12/0 (7.5/segment) and declines by apoptosis to 3.3 AMG/segment by stage 17. This reduction in number has been well-studied and a model proposed as follows (Bergmann et al., 2002). AMG begin to express a pro-apoptotic gene, head involution defective (hid) beginning at stage 13. The axon commissures secrete Spitz (Spi), which is an EGF-like protein. If sufficient Spi protein binds to Egfr located on the surface of AMG, MAP kinase is activated,which then phosphorylates and inactivates Hid, thereby blocking apoptosis. Thus, it is predicted that AMG in close proximity to the axon commissures will receive sufficient Spi to survive, and those not in close contact will undergo apoptosis (Wheeler, 2012).

Time-lapse imaging provided additional insight into AMG apoptosis. This process often occurred in a two-step manner. In segments with a large number of AMG (~10) there was a rapid wave of apoptosis that yielded ~5 AMG. Further reduction of AMG to ~3 cells occurred incrementally. In segments with a smaller number of AMG (~7), all AMG underwent apoptosis incrementally with no detectable apoptotic wave. Generally, the AMG that underwent apoptosis in initial waves were those at the anterior margin of the segment, furthest from the axon commissures. This is consistent with those cells receiving less axonal Spi. While all AMG cell death is likely due to inadequate Spi signal, time-lapse imaging experiments revealed instances in which AMG in close proximity to the commissures died, were cleared, and another AMG quickly assumed its position. Thus, even cells in apparent contact with axon commissures may not receive sufficient Spi for survival, suggesting additional complexity in the regulation of AMG apoptosis (Wheeler, 2012).

Previously it was argued that there might be a positive feedback loop in which Spi signaling from axons results in increased adhesion between AMG and axons. MG and axons adhere to each other via the heterophilic adhesion membrane proteins, Wrapper (on MG) and Neurexin IV (Nrx-IV) (on axons). Ensheathment and membrane projection into axons by AMG requires wrapper function. Expression of a wrapper enhancer is upregulated when Spi is provided ectopically. This suggests that Spi could promote stronger adhesion and more elaborate contacts between AMG and axons via increased Wrapper levels. Increased adhesion and surface area contact could expose AMG to higher concentrations of Spi making survival even more likely. In this way, AMG would compete for axonal interactions, with advantage going to those with more elaborate contacts (Wheeler, 2012).

There are ~4 PMG/segment at stage 12, and no evidence was found that PMG divide after their initial recruitment and specification by Notch and Hedgehog signaling. PMG number gradually declines to zero from stage 12/5 to stage 17. The PMG furthest from the PC die first. This suggests that the PMG adjacent to the PC may transiently receive a survival signal, although they too die between stages 16 and 17. It is generally thought that: (1) PMG do not share a large surface area with commissural axons (perhaps, in part due to low levels of Wrapper), (2) their survival cannot be rescued by high levels of Spi, and (3) their apoptosis is promoted by a different combination of proapoptotic genes than AMG. However, a detailed mechanistic analysis of PMG cell death has not been carried out, but is now feasible (Wheeler, 2012).

By combining staining of early embryos with time-lapse imaging of older embryos, it was possible to define in great detail the pattern of MG migration from stages 10 to 17. The AMG at stage 11 initially are present along the anterior/posterior axis in the anterior half of the segment and lie beneath (more external to) midline neurons and their precursors. The AMG nuclei begin to delaminate and migrate inward. Some AMG migrate internally and orient in a posterior direction toward the developing axon commissure. One possibility is that AMG initiate migration in response to and toward developing commissural axons or the MP1 neurons that lie just beneath the developing commissure. If axons provide guidance cues, migration may be due to an axon-derived secreted morphogen. If so, it is unlikely to be Spi, since in embryos in which both Egfr signaling is abolished and apoptosis inhibited, MG migration appears relatively normal. Another possibility is the PDGF- and VEGF-related factors (Pvfs) that may be secreted by axons or midline neurons and activates the PDGF and VEGF-related receptor (Pvr). Alternatively, MG could survey axonal membrane proteins at a distance using gliopodia, which are filopodia-like membrane extensions that act like growth cone filopodia. PMG also migrate inwards toward the developing axons and may be responding to similar guidance cues as AMG (Wheeler, 2012).

Not all AMG orient toward the developing axons. The anterior-most AMG elongate inward, but tend to orient toward the adjacent anterior segment before undergoing apoptosis. This suggests that they are either blocked from responding to the normal AMG guidance cues, possibly by other AMG, or are intrinsically different from the AMG that migrate in the posterior direction. Regarding intrinsic differences, most AMG-expressed genes analyzed at stages 11 and 12 are expressed in all AMG, and not in AMG subsets, indicating that obvious subsets of AMG are not apparent. One exception is the medial late-differentiating AMG present at stage 11 that are runt minus. However, after stage 11, these AMG are indistinguishable from other AMG, and it is uncertain whether their migratory paths are distinct. Consequently, it remains possible that different AMG subpopulations exist with intrinsic migratory differences, but there is currently little evidence to support this view (Wheeler, 2012).

Time-lapse imaging has provided new and definitive information regarding how individual MG acquire their positions surrounding the axon scaffold. One of the key observations was that the final migratory paths and positions of AMG correlated with their location at stage 12. AMG do not move randomly, they instead migrate in specific ways around the axon commissures to take their final positions. This suggests that AMG at different locations respond to specific, yet different, cues that guide them to their final positions. In addition, some mechanism must ensure that AMG cease their migration. The signals for both guidance and the termination of migrations might include a combination of AMGñAMG interactions, as well as interactions (either diffusible or contact-mediated) between AMG and different neurons, axons, or other substrata. As demonstrated by single-cell analysis, each AMG that ensheaths the commissures has extensive membrane projections that, in principle, can interact with other AMG and potential substrates. In both Nrx-IV and wrapper mutants, MG fail to adhere to the commissures and nearby neuronal cell bodies. However, AMG still migrate inward, migrate posteriorly to surround the AC, and maintain adhesion among themselves. This suggests that different adhesive interactions are required for different aspects of AMG migration (Wheeler, 2012).

The inward migration of MG suggests that there are 3 distinct groups of MG that may undergo collective guidance (AMG that migrate inward and anterior, AMG that migrate inward and posterior, and PMG that migrate inward and anterior). While each MG within these groups likely adheres to other group members, it is unknown whether the groups collectively signal to each other and whether they respond collectively or individually. However, after their initial inward migration, surviving AMG and PMG migrate to characteristic positions, indicating a transition to more individual behavior. Nevertheless, the surviving AMG remain in close contact with each other. Most importantly, this study provides the background for genetic screens that will identify and functionally analyze genes involved in AMG migration and commissure ensheathment (Wheeler, 2012).

Through the use of time-lapse and stained embryo imaging, the data presented in this paper provide a definitive descriptive view of MG migration. These results are consistent with and greatly extend earlier studies on MG development and migration. However, previous workers proposed detailed models regarding MG specification and migration, and, at this point, it is worth comparing our current view of MG development to earlier versions (Wheeler, 2012).

One influential and detailed model has a number of features: (1) there are 3 mature pairs of MG that ensheath the commissures, and are named MGA (anterior), MGM (middle), and MGP (posterior) based on their positions along the anterior/posterior axis, (2) these MG are derived from 3 discrete MG precursors that form prior to stage 8 and divide once yielding the 6 mature MG, (3) MGP migrate in an anterior direction into the next segment where they take up residence as the most posterior MG, (4) the MGM migrate above the AC and send projections that follow along the axons of the VUM neurons (medial tract), extending between the developing AC and PC, thus separating the commissures, and (5) the MGA migrate only a short distance, residing beneath the AC and PC. Important refinements of this model proposed later by other workers established that generally ~10 MG were generated, most underwent apoptosis, and that a group of posterior-residing MG underwent apoptosis and did not contribute to the ensheathment of the commissures. These posterior glia are equivalent to PMG in the current model (Wheeler, 2012).

With respect to the position, identity, and migration of the MGA,MGM, and MGP precursors and their progeny, previous studies employed lineage tracing of enhancer trap reporters reportedly from stage 16 to stage 8. However, since expression of the MG enhancer trap lines used does not begin expression until stage 12, tracing the origins of these cells back to stage 8 could not be directly determined, and was, presumably, conjectured. Subsequent work has demonstrated that MG do not form until at least stage 10 via Notch signaling, and that Hedgehog signaling at the same stage specifies AMG and PMG cell fates. Thus, the identities and positions of specific subtypes of MG cannot be identified before stage 10. Additionally, in previous studies no molecular markers were described that allowed MGA and MGM to be distinguished, so the proposed migration pathways that each of these MG took to reach their final destinations could not have been derived from stained samples (Wheeler, 2012).

More complicated are MGP and their relationship to PMG. In some respects, these cells appear equivalent. However, all PMG undergo apoptosis, and thus are not equivalent to MGP, which were initially proposed to survive and ensheath the PC. It is also noted that the PMG delaminate, migrate, and die within the same segment. No evidence was seen that they migrate from a more posterior segment (as proposed for MGP). It is possible that, in the absence of specific AMG and PMG markers, inward migrating AMG that orient toward the anterior were mistaken for migrating MGP (Wheeler, 2012).

Finally, regarding the issue of commissure separation, the previous model proposed that MGM tracked on the medial axon tract between the commissures, resulting in their separation. The possibility that MG may track along the medial tract to separate the commissures is an interesting idea, but has not been critically tested. This study found, from single-cell analysis, that both DM region and VM region AMG extend membrane/cytoplasm between the AC and PC, indicating that more than one AMG are usually positioned between the commissures. Together, these two cells ensure that the two commissures remain distinct and separate (Wheeler, 2012).

A global in vivo Drosophila RNAi screen identifies a key role of ceramide phosphoethanolamine for glial ensheathment of axons

Glia are of vital importance for all complex nervous system. One of the many functions of glia is to insulate and provide trophic and metabolic support to axons. Using glial-specific RNAi knockdown in Drosophila, this study silenced 6930 conserved genes in adult flies to identify essential genes and pathways. Among the screening hits, metabolic processes were highly represented, and genes involved in carbohydrate and lipid metabolic pathways appeared to be essential in glia. One critical pathway identified was de novo ceramide synthesis. Glial knockdown of lace, a subunit of the serine palmitoyltransferase associated with hereditary sensory and autonomic neuropathies in humans, resulted in ensheathment defects of peripheral nerves in Drosophila. A genetic dissection study combined with shotgun high-resolution mass spectrometry of lipids showed that levels of ceramide phosphoethanolamine are crucial for axonal ensheathment by glia. A detailed morphological and functional analysis demonstrated that the depletion of ceramide phosphoethanolamine resulted in axonal defasciculation, slowed spike propagation, and failure of wrapping glia to enwrap peripheral axons. Supplementing sphingosine into the diet rescued the neuropathy in flies. Thus, this RNAi study in Drosophila identifies a key role of ceramide phosphoethanolamine in wrapping of axons by glia (Ghosh, 2013).

Serine palmitoyltransferase catalyzes the condensation of serine and palmitoyl-CoA to generate 3-ketosphinganine, the rate-limiting step in de novo sphingolipid synthesis. Mutations in the two human subunits of the serine palmitoyltransferase are associated with hereditary sensory and autonomic neuropathy. A common feature of the mutations is the loss of canonical enzyme activity and the generation of toxic lipid intermediates. Glial inhibition of lace function (repo>mCD8-GFP/lace RNAi) resulted in glial bulging and in an alteration of the axonal packing in all eight pairs of abdominal nerves in all larval PNS examined. The bulging of glia was localized to focal regions, but appeared randomly along the entire peripheral nerves with diameters ranging from 10 μm to 30 μm. Nerves of repo-GAL4/+ control flies were straight and packed in bundles with a uniform diameter of 5-8 μm. In contrast, knockdown of lace in neurons did not result in any visible alterations of axonal morphology . The average cross-section area of the nerve were similar in the knockdown (elav-GAL4/lace RNAi) and in the elav-GAL4/+ control flies (Ghosh, 2013).

Notably, the ensheathment defect was not due to a compromised blood-nerve-barrier as has for example been observed in null fray mutants. In addition, the number of glial cells in the peripheral nerves was comparable to control. It is important to note glial cell death affects neuronal survival and results in embryonic lethality. The absence of embryonic lethality and the comparable glial cell number suggested that glial cell death did not occur at the larval stage after knockdown of lace. The expression of lace in glia was confirmed by double immunolabeling of lace5(LacZ enhancer trap line) with anti-β-galactosidase and anti-repo in L3 larval peripheral nerves. In addition, by RT-PCR analysis of the fly brain and PNS lace transcript was identified in the nervous system of both male and female flies (Ghosh, 2013).

Two independent RNAi lines (Transformant ID 21803 and 110181, VDRC) against lace showed identical swelling and wrapping defects. In addition, hypomorphic lace mutant (lace2/lace5) resulted in axonal defasciculation, ruling out off-target effects of the RNAi lines. As in the lace-RNAi knockdown, the average cross-section area of the nerves was increased in the hypomorphic lace mutant animals. The mutant phenotypes appeared to be subtle, which is not surprising as complete loss of lace during the development is lethal, while this hypomorphic combination are viable even into adulthood. However, 100% penetrance of the phenotype was observed both for the RNAi knockdownand the hypomorphic mutants. Importantly, the lace mutant phenotype was rescued by expressing UAS-lace specifically in the glial cells (repo-GAL4), pointing to an essential function of lace in glia (Ghosh, 2013).

Next, an analysis was performed to see in which of the different glial subtype lace was required. Glia subtype specific GAL4 drivers were used to silence lace function. A phenotype was only observed when lace was depleted in wrapping glia (Nrv2-GAL4). Quantification of GFP signal intensity revealed that membrane area was significantly reduced as compared to control (Nrv2>mCD8GFP/lace RNAi versus Nrv2>mCD8GFP). Similar results were observed when the Nrv2>mCD8-mcherry driver line was used to knockdown lace in the wrapping glia. In contrast, knockdown of lace in the two other glial subtypes, the subperineurial (gliotactin-GAL4) and the perineurial (NP6293-GAL4), did not lead to any visible changes (glial swellings or decrease in the GFP signal intensity) in glia or in axons (-G), suggesting a predominant role of lace in the encapsulation of peripheral nerves (Ghosh, 2013).

To examine the ultrastructure in more detail electron microscopy was performed. Electron micrographs clearly showed that knockdown of lace in glia (repo/lace RNAi) severely impaired axonal enwrapping compared to control (repo/+). Notably, also in the non-swollen regions (A2-A3 segment) of the nerve much less glial processes covered the axons. Quantification demonstrated a significant increase in the number of completely unwrapped axons in this region. A similar phenotype was observed when lace was knocked down in wrapping glia (Nrv2/lace RNAi). Again, a clear increase in the completely unwrapped axons was detected as compared to the controls. Importantly, TUNEL assay could not detect any apoptotic glial nuclei (Nrv2>laceRNAi) suggesting that loss of axonal ensheathment is not because of dying wrapping glial cells. Together, these results indicate that sphingolipids or intermediates of the sphingolipid pathway are necessary for membrane expansion of wrapping glia (Ghosh, 2013).

In order to search for the specific sphingolipid (SL) species required by the wrapping glia to mediate axonal ensheathment, a genetic dissection study was performed by expressing RNAi against all known SL metabolic enzymes selectively in glia. Out of 12 genes, knockdown of Spt-I, schlank, Des1 and Pect in glia (repo>mCD8-GFP/RNAi) with two different RNAi lines (except Des1 due to unavailability) phenocopied the glial swelling and axonal defasciculation as observed upon loss of lace function. Interestingly, all four genes that show 100% penetrance are known to be involved in the biosynthesis of ceramide-phosphoethanolamine (CerPE). The specificity of the effect was demonstrated by the absence of any visible phenotype after neuronal specific knockdown of Spt-I, schlank, Des1 and Pect. Additionally, glial specific knockdown of different ceramide derivative synthesizing enzymes (GlcT1, CGT, CerK) and PE synthesizing enzyme (bbc) did not show any visible defects of axon or glial morphology. Moreover, when Spt-I, schlank, Des1 or Pect were knocked down specifically in wrapping glia, wrapping defects similar to the lace phenotype were observed . The quantification revealed that the GFP signal intensity was significantly reduced in all four experiments as observed after lace knockdown. Ultrastructural analysis by transmission electron microscopy also showed that wrapping glia failed to extend their membrane around the axons; and consequently there was an increase of the completely unwrapped axons. Hence, the data strongly suggests an essential function of glial CerPE in axonal ensheathment by wrapping glia (Ghosh, 2013).

In order to analyze whether knockdown of lace, schlank, Des1 and Pect resulted in depletion of CerPE levels, a detailed lipidomics analysis of the nervous system was performed. This is particularly important in RNAi studies targeting enzymes, because residual enzyme activity due to inefficient RNAi-silencing is often sufficient for their function. Since the nervous system of Drosophila only contains 10% of glia, the RNAi was expressed both in neurons and glia using repo-GAL4 and elav-GAL4 drivers to deplete the enzymes in the entire nervous system. L3 larval brain and peripheral nerves were dissected and lipidomics analysis was performed with high-resolution shotgun mass spectrometry. Importantly, lipidomics analysis confirmed that knockdown of lace, schlank, Des1 and Pect reduced CerPE levels significantly, whereas triacylglycerol (TAG) and diacylglycerol (DAG) and sterol levels were unaltered. Ceramide levels were reduced upon downregulation of lace and Des1, whereas knockdown of Pect lead to increased ceramide levels consistent with its function as a phosphoethanolamine cytidylyltransferase. Phosphatidylcholines (PC) and Phosphatidylethanolamine (PE) levels were slightly changed possibly due to compensatory mechanisms (Ghosh, 2013).

Next, tests were performed to see whether it was possible to rescue the morphological phenotype induced by knockdown of lace in glia by supplementing sphingosine (re-converted to ceramide by condensation with a fatty-acylCoA catalyzed by the various ceramide synthases) into the diet of the flies. Indeed, the phenotype of glia-specific knockdown of lace was efficiently rescued by the exogenous addition of sphingosine (300 μM) to the food. Double Immunolabelling of glia and neuronal membrane reveals that the glial bulging and axonal unpacking was rescued upon addition of sphingosine to the diet. Orthogonal projections and the quantification demonstrated the rescue of the neuropathy like phenotype in flies. It was furthermore observed with the ultrastructural analysis that the glial enwrapment defect was recovered upon sphingosine addition to the diet. Quantitative analysis of the peripheral nerves using the confocal and electron microscopy showed that the oral administration of sphingosine can restore the enwrapping defect and the neuropathy-like phenotype (Ghosh, 2013).

Sphingolipids have both structural and signalling functions in cells. CerPE is a relatively low abundant lipid constituting only around 1% of the total fly lipidome. Interestingly, CerPE appears to be enriched in the fly brain membrane lipidome (4%). In mammals, CerPE is only found in trace amounts, since sphingolipids are in general built on ceramide phosphatidylcholine in higher organisms. There are different possibilities of how CerPE could exert its function in glia. CerPE might be required for signal transduction pathways that control membrane synthesis in wrapping glia. Recently, a mutation in egghead, an enzyme that extends the glycosphingolipids (GSLs) in flies, causes the proliferation and overgrowth of subperineurial glia mediated by aberrant activation of phosphatidylinositol 3-kinase-Akt pathway. CerPE may also increase the packing density of the lipids in the membrane, thereby helping to build up an efficient barrier for the electrical insulation of the axons. In vertebrates, a related sphingolipid, galactocylceramide, is critical for the formation of an insulating myelin sheath in oligodendrocytes. Galactosylceramide and/or its sulphated form are required for the tight sealing of the glial paranodal membrane to the axon. Interestingly, mice lacking ceramide synthase 2, a vertebrate homolog of schlank, have myelination defects (Ghosh, 2013).

Alterations of enzyme function or enzyme deficiencies do not only result in a reduction in the amount of an essential product, but can also lead to the accumulation of a toxic intermediate, or the production of a toxic side-product. For example, mutations in human serine palmitoyltransferase result in a loss of normal enzyme function causing a shift in the substrate specificity, which increase the accumulation of atypical, toxic lipid products. Thus, gain-of-toxic-function is another possibility of how knockdown of lace may cause the axonal ensheathment defects (Ghosh, 2013).

Interestingly, supplementing sphingosine to the diet restored the ability of wrapping glia to extend their membrane around axons. How diets affect the distribution of lipids in cells and thereby modulate biological processes will be an important question for future investigations. Drosophila is an ideal system to pursue such studies because of the short life span and the powerful genetics, which enable rapid and detailed analysis. In summary, the current study illustrates that a large-scale screen in Drosophila, in combination with concomitant morphological and electrophysiological analysis has the potential to dissect the basic mechanisms of neuron-glia communication. Detailed knowledge of neuron-glia interactions is a pre-requirement for the rational design of treatment strategies for neuropathies or other diseases in the future (Ghosh, 2013).

Gliopodia extend the range of direct glia-neuron communication during the CNS development in Drosophila

Midline glia are a source of cues for neuronal navigation and differentiation in the Drosophila CNS. Despite their importance, how glia and neurons communicate during development is not fully understood. This study examined dynamic morphology of midline glia and assessed their direct cellular interactions with neurons within the embryonic CNS. Midline glia extend filopodia-like 'gliopodia' from the onset of axogenesis through the near completion of embryonic neural development. The most abundant and stable within the commissures, gliopodia frequently contact neurites extending from the neuropil on either side of the midline. Misexpression of Rac1N17 in midline glia not only reduces the number of gliopodia but also shifts the position of neuropils towards the midline. Midline-secreted signaling protein Slit accumulates along the surface of gliopodia. Mutant analysis supports the idea that gliopodia contribute to its presentation on neuronal surfaces at both the commissures and neuropils. It is proposed that gliopodia extend the range of direct glia-neuron communication during CNS development (Vasenkova, 2005).

Axonal and dendritic growth cones are guided into commissures, longitudinal fascicles and neuropils within the embryonic CNS. Throughout this period, midline glia extend gliopodia not only into commissures near the midline but also at the medial region of longitudinal neuropils several cell diameters away from their edges. While navigating within the CNS, neuronal growth cones also extend filopodia that span several cell diameters. Put together, the distance of direct filopodial contact between midline glia and neurons expands dramatically. As a result, the embryonic CNS displays a 'filopodial web' in which a majority of neurons could contact midline glia directly. The main effect of gliopodial presence during CNS development could be to extend the reciprocal influence of specialized midline cells over, and/or sensitivity toward, outgrowing neurites that lie at a distance. Whether or not a similarly extensive 'filopodial web' exists in embryonic nervous systems of larger animals is yet to be determined. However, it is interesting to note that the floorplate cells at the midline in the vertebrates have been documented to extend 'side-arms' (Vasenkova, 2005).

Midline glia in Drosophila are generally thought to serve a role analogous to floorplate cells of the vertebrate CNS. Unlike other glia that migrate along the medial-lateral axis of the embryonic nervous system, midline glia remain at the midline of the CNS throughout development. Yet, the distance over which midline glia-derived factors exerts their influence reaches at least the neuropil. Although a subset of axons and dendrites cross the midline through commissures, the remaining majority stays within the neuropil. Axons from the peripheral sensory neurons terminate here as well, without crossing the midline. The positioning of these CNS neurites and PNS terminals is apparently under the influence of Slit, a midline glia-derived signal protein. This study shows that midline signaling protein Slit is enriched extracellularly along gliopodia. Thus, Slit, which has been previously thought to reach neuropil through diffusion, could be also 'hand-delivered' (or foot-delivered?) onto the neuropilar surface by gliopodia (Vasenkova, 2005).

Slit is perhaps the easiest endogenous midline-derived signaling molecule to be examined for its redistribution in vivo through immunocytochemistry. This is because in the CNS, unlike other molecules such as Netrins that are also expressed by surrounding neurons, Slit is exclusively supplied by midline glia. In theory, however, other midline glia-derived molecules could also be carried through gliopodia. Vesicular transport of synaptic vesicle proteins has been observed along neuronal filopodia. Similarly, vesicles containing midline-signaling molecules could travel along gliopodia to neuropils where simple diffusion of the cue might not be effective or sufficiently discrete to mediate cell-specific interactions. The data suggest that gliopodia could serve as a vehicle for cue delivery to axons and dendrites that develop within commissures and neuropil of the embryonic CNS (Vasenkova, 2005).

In addition to delivering secreted molecules, gliopodia could also allow membrane-associated molecules to reach over a long distance from their source. Through dynamic gliopodial behavior, even membrane-bound ligands could create a local gradient of concentration, or availability, similar to the situation with diffusible ligands. However, unlike diffusible molecules, such molecules convey information regarding the identity of cells that produce them by virtue of being integrated into that cell's membrane. They can therefore serve as cell-recognition molecules that neurons detect along the whole length of gliopodia. In contrast, some gliopodia-bound molecules can also serve as remote-sensing receptors for the glia. Thus, discrete two-way communication through cell-bound molecules, often thought of as being an exclusive privilege of short-distance cellular encounters, is possible even for long-distance communication among glia and neurons. This extended molecular communication through the “filopodial web” could bear large impact on the course of embryogenesis in which the initial size of its emerging nervous system is small (Vasenkova, 2005).

Distinct types of glial cells populate the Drosophila antenna

The development of nervous systems involves reciprocal interactions between neurons and glia. In the Drosophila olfactory system, peripheral glial cells arise from sensory lineages specified by the basic helix-loop-helix transcription factor, Atonal. These glia wrap around the developing olfactory axons early during development and pattern the three distinct fascicles as they exit the antenna. In the moth Manduca sexta, an additional set of central glia migrate to the base of the antennal nerve where axons sort to their glomerular targets. In this work, whether similar types of cells exist in the Drosophila antenna has been investigated. Different P(Gal4) lines were used to drive Green Fluorescent Protein (GFP) in distinct populations of cells within the Drosophila antenna. Mz317::GFP, a marker for cell body and perineural glia, labels the majority of peripheral glia. An additional ~30 glial cells detected by GH146::GFP do not derive from any of the sensory lineages and appear to migrate into the antenna from the brain. Their appearance in the third antennal segment is regulated by normal function of the Epidermal Growth Factor receptor and small GTPases. These distinct populations of cells have been denoted as Mz317-glia and GH146-glia respectively. In the adult, processes of GH146-glial cells ensheath the olfactory receptor neurons directly, while those of the Mz317-glia form a peripheral layer. Ablation of GH146-glia does not result in any significant effects on the patterning of the olfactory receptor axons. This study has demonstrated the presence of at least two distinct populations of glial cells within the Drosophila antenna. GH146-glial cells originate in the brain and migrate to the antenna along the newly formed olfactory axons. The number of cells populating the third segment of the antenna is regulated by signaling through the Epidermal Growth Factor receptor. These glia share several features of the sorting zone cells described in Manduca (Sen, 2005).

Studies in Manduca, have described three types of glia associated with the developing olfactory neuropil and nerve: (1) peripheral glia that arises from the antenna and ensheath olfactory axons; (2) lobe neuropil glia that surround and stabilize olfactory glomeruli; (3) sorting zone glia which are central in origin and migrate to the base of the antennal nerve where they serve to segregate axons that target independent glomeruli. In Drosophila, previous work has described the direct equivalents of peripheral antennal and the neuropil glia. This paper identifies an addition set of glia marked by GH146::GFP that share common features with sorting zone glia. A model suggests that these glia arise in the brain and migrate along the ORNs which have newly connected to the brain. Cells proliferate as they migrate along the nerve and enter the third segment of the antenna. Coincident with GH146-glia arrival at the periphery, the Ato-derived glia marked by Mz317::GFP (that are already present on the olfactory axons) move to wrap around the cell bodies of the sensory organs. The GH146-glial processes tightly ensheath the olfactory fascicles and form a layer below that of the Mz317-glial projections. In all 36 hrs APF preparations examined, ~30 GH146-glia were detected suggesting there must exist mechanisms to regulate the number of cells terminating in the antenna (Sen, 2005).

What is the function of GH146-glia during olfactory development? Although these cells share many of the properties of sorting zone glia described in the moth, it was not possible to implicate them in any guidance function. In Manduca, Fasciclin II (FasII), the insect ortholog of N-CAM, is expressed in a subset of ORNs scattered throughout the antennal nerve. Upon reaching the glial rich sorting zone, these projections segregate from the non-expressing axons and terminate in distinct glomeruli. In Drosophila, the presence of FasII protein on the ORNs during axon projection could not be detected using the available antibodies. When the GH146-glia were completely absent within the third antennal segment, no significant abnormalities were detected in ORN fasciculation within the antenna. The processes of the GH146-glia extend into the antennal nerve to ensheath axon bundles. This segregation is not based on Or gene type and its functional significance is obscure. A similar migration of glial cells from the centre into the periphery has been described during development of the Drosophila compound eye (Sen, 2005).

What are the factors that induce migration of glia from the brain towards the periphery? Cell migration in insect glia is well known to be mediated by signaling through the Fibroblast Growth Factor Receptors (FGFRs). Preliminary observations appear to rule out a requirement for FGFR signaling during GH146-glial migration. The downstream effector of FGF (Dof), is not expressed in these cells at any time during their development. Further, expression of dominant negative forms of Drosophila FGF receptors does not affect glial cell number in the antenna (Sen, 2005).

It is suggested that GH146-glial migration is mediated by EGF signaling. The ligand for Egfr, Vein, has been shown to be known to be expressed in the antennal epidermis at a time consistent with the arrival of cells from the center. The mechanisms involved in triggering this homing of cells into the antenna require extensive investigation (Sen, 2005).

Identity, origin, and migration of peripheral glial cells in the Drosophila embryo

Glial cells are crucial for the proper development and function of the nervous system. In the Drosophila embryo, the glial cells of the peripheral nervous system are generated both by central neuroblasts and sensory organ precursors. Most peripheral glial cells need to migrate along axonal projections of motor and sensory neurons to reach their final positions in the periphery. This paper studied the spatial and temporal pattern, the identity, the migration, and the origin of all peripheral glial cells in the truncal segments of wildtype embryos. The establishment of individual identities among these cells is reflected by the expression of a combinatorial code of molecular markers. This allows the identification of individual cells in various genetic backgrounds. Furthermore, mutant analysis of two of these marker genes, spalt major and castor, reveal their implication in peripheral glial development. Using confocal 4D microscopy to monitor and follow peripheral glia migration in living embryos, it was shown that the positioning of most of these cells is predetermined with minor variations, and that the order in which cells migrate into the periphery is almost fixed. By studying their lineages, the origin of each of the peripheral glial cells was uncovered and they were linked to identified central and peripheral neural stem cells (von Hilchen, 2008).

This study has characterized the expression of a collection of cell-specific molecular markers, which allows to identify and distinguish all glial cells in the embryonic peripheral nervous system. The reproducibility with which enhancer-trap lines and marker genes are expressed in the individual peripheral glial cells, indicates that these cells display unique identities. Furthermore, the spatial and temporal pattern of migration and the final arrangement of these cells are relatively stereotypic. This suggests that the specification of the unique identity of each cell does not only define a specific combination of genes to be expressed, but also includes the information about the timing of migration, the nerve tract it is associated with, and to some degree the final position to be occupied along the respective nerve. How the cell receives this information is still unknown. The individual characteristics could be determined (1) by lineage or (2) during migration by cell-cell interactions (between the glial cells or between the glia and other closely associated cells, e.g. neurons, tracheae), or (3) by a combination of both (von Hilchen, 2008).

The master regulatory gene glial cells missing (gcm) is required to induce the glial cell fate. Gcm as a transcription factor switches on downstream target genes, of which the gene encoding for the homeobox transcription factor Reversed polarity (Repo) is well described. As this cascade of gene activation is required for all glial cells in the Drosophila embryo (except the midline glia), it is unlikely to contribute to cell fate diversification among the glia. Whereas central glial cells migrate over rather short distances, in literally any possible direction, to finally occupy stereotypic positions within the CNS, the peripheral glial cells behave differently as they have to migrate over remarkable distances into the periphery. It has been recently shown that the migration of PGs depends on Notch signalling. In Notch mutants or in mutants where Notch signalling is altered in PGs, the migration is impaired or even completely blocked. However, this signalling does not appear to supply the cells with characteristics of their fate apart from the onset and/or maintenance of the migration itself. Sepp (2000) described the developmental dynamics and morphology of a subset of peripheral glial cells and could show that a signalling cascade mediated by the small GTPases RhoA and Rac1 influences the actin cytoskeleton of migrating PGs. Sepp further showed, that, within the analysed population of cells, a 'leading glia’ expresses filopodia-like structures whereas the ‘follower’ cells do not. Similar results were reported by Aigouy (2004). Aigouy established a 4D microscopy technique to record and analyse the developmental dynamics and migratory behaviour of PNS glia during pupal stages in the developing fly wing. In this system, differences between 'leading' and 'follower' glia cells were also observed. The glial cells in the wing PNS migrate along wing veins in a chain with one 'leading' cell in front. If this chain is interrupted by laser ablation of either the leading or intermediate cells, a new 'leading’ cell starts to form filopodia and explores the surrounding. Once this new 'leading' cell catches up with the previous chain or reaches its target area, the filopodia disappear and the cells' morphology changes again. Hence, these differences in glial cell morphology and behaviour in the wing PNS are based on interactions of the glial cells with each other rather than on a predetermined intrinsic cell fate (von Hilchen, 2008).

Findings for the embryonic PNS glia suggest that these cells are predetermined at least to a certain extent. The 4D microscopy approach allowed tracing of the migration of individually identified PGs in living embryos from the moment they leave the CNS until they reach their final position. Apart from the dorsal SOP-derived cells, which never change their position or behaviour, it is always the ePG9 that leaves the CNS first and 'leads' the track. This cell expresses filopodia-like structures, while the following cells do not, although it remains to be experimentally shown whether they can take over the leading function in the absence of ePG9. It is worth mentioning that the SOP-derived ePG12 migrates along trajectories of the ISN prior to ePG9. It is not clear whether ePG12 has any leading function for ePG migration or functions as a guidepost cell for axonal projections. It is the only cell, though, that swaps nerve tracts and finally associates with the TN. Most likely, cell-cell communication between ePG12 and axonal projections and/or neighbouring cells is required for proper pathfinding and positioning. It is always the ePG4 that migrates along and finally enwraps the segmental nerve. As this cell is the only cell associated with the distal part of the segmental nerve, it functions as 'leading' glia for this nerve and expresses filopodia-like structures at least in later stages when it enwraps the SN. This enwrapment occurs in a bidirectional fashion, i.e. the filopodia occur at both ends of the glial cell (von Hilchen, 2008).

Lineage analysis revealed that the PGs mentioned above originate from the CNS neuroblast NB 1-3 and a ventrally located SOP. Interestingly, the two NB 2-5 derived PGs (ePG6 and ePG8) differ from these cells with respect to both identity and behaviour. They express fewer of the analysed PG-specific markers (cas-Gal4 and mirr-lacZ) and it is not possible to distinguish between these two cells so far. Whether the lack of identifying markers is a consequence of or a prerequisite for their different identity and behaviour is not yet clear. The cells migrate along the ISN independently of the NB 1-3- and SOP-derived PGs and frequently overtake them (and occasionally even one another). The correlation of such characteristics with the different origin of these three subpopulations of PGs lends support to the hypothesis that some aspects of cell fate diversification among the PGs may be predetermined by lineage. It is likely, that such predetermined characteristics include the competence to respond to specific external signals that guide the respective cell along the correct nerve to its target position (von Hilchen, 2008).

One incidence for lineage-dependent cell fate determination comes from the analysis of the ladybird homeobox genes. The ladybird genes are expressed in the developing CNS in only few NBs including NB 5-6. The NB 5-6 lineage produces one of the proximal PGs (ePG2) which expresses the Ladybird early (Lbe) protein. It has been shown that a loss of ladybird gene function results in a loss of the ePG2 in a third of all analysed hemisegments, accompanied with a higher number of medially located glial cells in the CNS. An opposite phenotype with excessive cells in the transition zone was observed by ectopic expression of the ladybird genes throughout the CNS. Using an anti-Repo antibody as well as a subset specific reporter transgene (K-lacZ), De Graeve (2004) provided evidence suggesting that the ladybird genes play a role in glial subtype specification in particular NB lineages. Another factor shown to be required for the specification of a lineage-specific set of glial cells (NB1-1-derived subperineurial glia) is Huckebein, which interacts with Gcm to amplify its expression specifically in these cells (von Hilchen, 2008).

Furthermore, in cas mutants, it was shown that the two NB 2-5-derived glia (ePG6 and ePG8) do not migrate into the periphery but most likely stay at their place of birth, although they acquire glial cell fate (as can be deduced from Repo stainings). Thus, similar to Ladybird and Huckebein, Cas seems to be involved in lineage-dependent glial subtype specification rather than determination of glial fate in general. In contrast to ladybird (De Graeve, 2004), though, Cas is not sufficient to ectopically induce glial cell fate or PG subtype specification (von Hilchen, 2008).

This study shows that salm is a likely candidate participating in the control of glial development. Embryos homozygous for salm445 show a pleiotropic and variable phenotype affecting not only glial cells but also PNS neurons, sensory organs, and other tissues. Yet, nearly all ventrally derived PGs stall in the transition zone between CNS and PNS and do not migrate properly into the periphery. In about 50% of the analysed hemisegments, a variable number of one to three PGs are missing, even though these cells could remain in the CNS. salm-lacZ is expressed in the two ventral SOP-derived ePG4 and ePG5, as well as in the dorsal SOP-derived ePG11 along the DLN, and in some of the ligament cells of the lateral chordotonal organ. In salm445 mutants the ePG4 cell can sometimes be detected at its wildtypic position along the SN. If ePG4 is missing along the SN, it could well be a secondary effect, as the SN itself is affected with the SNc shortened or occasionally missing. The ePG5 however, cannot be unambiguously identified in Repo-staining within the group of cells stalling in the transition zone (von Hilchen, 2008).

It needs to be further shown whether the differences between the PGs derived from certain progenitor cells result in functional differences in the larva. The peripheral nerves of the larva are ensheathed by two distinct types of glial cells, the perineurial and the subperineurial glial cells. The subperineurial glia build septate junctions with each other (or themselves) and thereby form the blood-nerve barrier, whereas the perineurial glia form an outer layer and secrete the neural lemma. In order to allow proper electrical conductance, the peripheral nerves must be enwrapped and insulated at the end of embryogenesis when hatching of the larva requires coordinated muscle contractions. It is not known to date which of the embryonic PGs will become perineurial or subperineurial glia, or what other functions they might fulfill (von Hilchen, 2008).

The comprehensive description of the ancestry, identity and dynamics of the developing embryonic peripheral glia, and the molecular markers at hand, provide a crucial basis for further clarification of the mechanisms controlling development, migration, and function of peripheral glia on a single cell level (von Hilchen, 2008).

Organization and postembryonic development of glial cells in the adult central brain of Drosophila

Glial cells exist throughout the nervous system, and play essential roles in various aspects of neural development and function. Distinct types of glia may govern diverse glial functions. To determine the roles of glia requires systematic characterization of glia diversity and development. In the adult Drosophila central brain, five different types of glia were identified based on its location, morphology, marker expression, and development. Perineurial and subperineurial glia reside in two separate single-cell layers on the brain surface, cortex glia form a glial mesh in the brain cortex where neuronal cell bodies reside, while ensheathing and astrocyte-like glia enwrap and infiltrate into neuropils, respectively. Clonal analysis reveals that distinct glial types derive from different precursors, and that most adult perineurial, ensheathing, and astrocyte-like glia are produced after embryogenesis. Notably, perineurial glial cells are made locally on the brain surface without the involvement of gcm (glial cell missing). In contrast, the widespread ensheathing and astrocyte-like glia derive from specific brain regions in a gcm-dependent manner. This study documents glia diversity in the adult fly brain and demonstrates involvement of different developmental programs in the derivation of distinct types of glia. It lays an essential foundation for studying glia development and function in the Drosophila brain (Awasaki, 2008).

Two layers of distinct glial cells surround the entire adult Drosophila brain. The inner one, possibly composed of a fixed number of large sheet-like subperineurial glial cells since larval hatching, constitutes the fly blood-brain barrier in both larval and adult brains. In contrast, the outer layer consists of numerous small oblong perineurial glial cells that proliferate through postembryonic development of the brain and may remain immature until formation of the adult brain. Given its more external location and apparent adherence to the subperineurial layer, the perineurial glia possibly helps make up the adult blood-brain barrier. However, these two types of surface glia likely play nonoverlapping, complementary roles in the regulation of the permeability to the CNS, as implied from their different enhancer trap expression patterns (Awasaki, 2008).

The other three types of glia form independent networks inside the brain. Cortex glia enwrap individual neuronal cell bodies in the brain cortex. Each cortex glial cell send mesh-like processes to surround multiple neuronal cell bodies locally. Cortex glia may provide metabolic support for neurons and have the potential for modulating neuronal cell body functions in a spatially controlled manner. Further inside the brain lie two types of neuropil glia. The ensheathing glia outlines individual neuropils and their subcompartments, while the astrocyte-like glia extend processes into the neuropils where synapses form (Awasaki, 2008).

The glial boundary established by the ensheathing glia may provide the necessary insulation to prevent lateral communication between adjacent neuropils, and provide septa when a neuropil is composed of multiple independent subcompartments. In contrast, astrocyte-like glial cells potentially associate with synapses. Several electron microscopic studies have shown that glial processes are located around synapses of insect neuropils. In an analogous manner, mammalian astrocytes target processes to synapses. They express excitatory amino-acid transporters (EAAT-1 and -2), and can recover excess extracellular glutamate or aspartate to keep the local environment suitable for the next synaptic transmission. In addition, mammalian astrocytes may release gliotransmitters in response to synaptic activity to modulate synaptic functions. Whereas in the vertebrate nervous system synapses are formed not only on the dendrites and axon terminals but also on the surface of the neural cell bodies, in the invertebrate brain no synapses are formed on the neuronal cell bodies. Accordingly, the cortex glia and neuropil glia are associated exclusively to the neural cell bodies and synaptic neuropils, respectively. Although Drosophila glia also expresses EAAT-1 (dEAAT-1) that is concentrated in neuropils, it remains to be determined which function of the vertebrate astrocytes is achieved by which type of insect glial cells (Awasaki, 2008).

In the adult brain or ventral nerve cord of other insects, two types of glial cells associated with neuropil have been identified; type II and type III glia or interface and neuropilar glia in Musca, interface and neuropil glia in Acheta and neuropil cover type and neuropil glia or simple and complex neuropil glia in Manduca. One type is localized cortex/neuropil interface or borders among neuropils and ensheaths neuropil, and another type extends glial processes inside the neuropils, like the ensheathing and astrocyte-like glia of adult Drosophila brain, respectively. It is, therefore, these two subtypes of neuropil glia would commonly exist in insects (Awasaki, 2008).

These distinct types of adult fly brain glia arise independently from each other. The proliferation of subperineurial glia is probably restricted to embryogenesis, and any postembryonic increase in the cortex glial cell number should be much less significant than the expansion of the other three types of adult glia, which primarily occurs during larval development. Furthermore, distinct populations of precursors give rise to perineurial, ensheathing, or astrocyte-like glia. Notably, perineurial glia precursors distribute around the brain surface and make clones that expand locally as the brain grows in size through development. In contrast, ensheathing and astrocyte-like glial cells are derived from specific proliferation centers and do not migrate throughout the brain until metamorphosis. Most somatic clones of neuropil glia exclusively consist of ensheathing or astrocyte-like glial cells. However, the identity of the neuropil clones could not be unambiguously determined until late pupal stages, making it unclear whether the progenitors of the ensheathing and astrocyte-like glia are mixed in those proliferation centers. Perineurial glial precursors make clones of similar sizes at a given developmental stage, suggesting that they retain the same proliferation potential through development. In contrast, the clones of neuropil glia derived at the same stage vary in size, raising the possibility that neuropil glial precursors undergo stochastic proliferation, possibly, in response to some limited local cue(s). Supply of Vein, a neuregulin-like trophic factor, from neurons has been shown to promote local proliferation of the embryonic longitudinal glia. It remains to be determined whether similar mechanisms govern the postembryonic expansion of the adult neuropil glia. Finally, all the postembryonic glial precursors apparently divide symmetrically to produce two daughters following each division; intriguingly, the final round of mitosis consistently takes place around pupal formation. This suggests a stage-specific signal may trigger the final glia-producing mitoses (Awasaki, 2008).

The involvement of different developmental programs in the derivation of different types of adult glia is further evidenced in the differential temporal requirement for gcm. GCM promotes all embryonic gliogenesis except for midline glia. GCM suppresses neural differentiation through expression of Tramtrack and confers glial identity via induction of Repo and other glial genes. Transient expression of gcm also occurs in the postembryonic development of both medulla and thoracic glial lineages, and inhibition of GCM function by expression of a dominant-negative form of GCM suppresses differentiation of their glial development. In addition, this study shows that precursors of neuropil glia express gcm transiently and the postembryonic induction of the gcm mutant clone effectively arrest the development of neuropil glia. These observations indicate that GCM plays analogous roles in promoting glial fate at different developmental stages. It is possible that the precursors of neuropil glia do not acquire full glial identity until gcm is expressed at an intermediate stage of development. In contrast, gcm is dispensable for postembryonic development of perineurial glia. Unlike midline glia in which Repo is never expressed, perineurial glial cells are strongly positive for Repo, a direct target for GCM, making it likely that GCM is involved in the development of perineurial glia. It appears more plausible that like neuropil glia transient expression of gcm also occurs in the perineurial glial precursors but at an earlier stage, and that they have already acquired glial cell fate before larval hatching (Awasaki, 2008).

While most glial types appear to be conserved in flies, the microglia type of glia is not obvious in the adult fly brain. It is possible that not all major types of glia were identified because of the restriction of of the analysis to only Repo-positive cells. Nevertheless, evidence is accumulating that neuropil glia may share microglia-type functions to engulf degenerating axons in the process of programmed axon pruning or following injury of neurites. Additional glial diversity has been suggested in vertebrates. Many subtypes of glial cells in the Drosophila embryonic ventral ganglion has also been revealed through detailed analysis of lineage as well as gene expression. The identification of five major types of adult fly brain glia and the analysis of their developmental origins pave a way for further use of the Drosophila as a model system for study of glial diversity, development, and function (Awasaki, 2008).

miR-31 mutants reveal continuous glial homeostasis in the adult Drosophila brain

The study of adult neural cell production has concentrated on neurogenesis. The mechanisms controlling adult gliogenesis are still poorly understood. This study provides evidence for a homeostatic process that maintains the population of glial cells in the Drosophila adult brain. Flies lacking microRNA miR-31a start adult life with a normal complement of glia, but transiently lose glia due to apoptosis. miR-31a expression identifies a subset of predominantly gliogenic adult neural progenitor cells. Failure to limit expression of the predicted E3 ubiquitin ligase, Rchy1, in these cells results in glial loss. After an initial decline in young adults, glial numbers recovered due to compensatory overproduction of new glia by adult progenitor cells, indicating an unexpected plasticity of the Drosophila nervous system. Experimentally induced ablation of glia was also followed by recovery of glia over time. These studies provide evidence for a homeostatic mechanism that maintains the number of glia in the adult fly brain (Foo, 2017a).

Despite the critical role that glia play in the proper functioning of neurons, study of adult neural cell production has focused mainly on neurons. Much less is known about gliogenesis in the adult brain. This report provide several lines of evidence for ongoing gliogenesis in the adult. These data include labelling the newly synthesized DNA of newly born cells in the adult showed the production of glia as well as neurons. Genetic mosaic analysis to produce clones of marked cells in the adult brain showed the production of new neurons and new glia from progenitor cells expressing Insc-Gal4 as well as a newly identified population of progenitors that express the miR-31a microRNA. Interestingly, the miR-31a-expressing progenitors appear to be mainly gliogenic, while those expressing Insc-Gal4 are mainly neurogenic. Given that both progenitors can make neurons and glia, it is suggested that the miR-31a-expressing progenitor cells may be a specialized subset of Insc-Gal4-expressing neuroglial progenitors. This is supported by the observation of miR-31a sensor activity in a subset of Insc-Gal4 cells (Foo, 2017a).

In the absence of miR-31a expression, glial number is affected. It is noted that the number of glia is normal in young adult miR-31a mutants, drops sharply over several days and then recovers. This pattern implies an active process of glial turnover in the young adult brain, which is impacted by the absence of the miRNA in the progenitor population. Evidence is provided that this defect is due to the requirement for the miRNA in progenitor cells, but not in mature glia or neurons (Foo, 2017a).

The findings show that miR-31a acts through regulation of Rchy1, a predicted E3 ubiquitin ligase. Although many transcripts are misregulated in the mutant, restoring expression of Rchy1 towards normal levels effectively suppressed the glial defect in the mutant. This implies that the overexpression of Rchy1 contributes to the failure of these cells to produce sufficient glia during the remodelling process that was observed in the young brain. In miR-31a mutants, Rchy1 protein was detected at elevated levels in the progeny of the miR-31a progenitor cells. Excess Rchy1 appears to be detrimental to the survival of the glial progeny, in that loss of glia can be suppressed by blocking apoptosis. There was only a small change in the number of neurons in adult brains of miR-31a mutants compared to wild-type animals. But, it is noted that the miR-31a progenitor cells are few in number compared to the predominantly neurogenic Insc-Gal4-expressing progenitors. Therefore, an effect of excess Rchy1 in these cells on production of neurons cannot be ruled out (Foo, 2017a).

Ubiquitin-mediated protein turnover has been shown to play several important roles during CNS development in Drosophila. Ubiquitination and proteosomal degradation of glial cells missing allow embryonic glia to exit the cell cycle and begin differentiation. Additionally, neural cell fate can be controlled by ubiquitin-dependent regulation of protein translation. This study shows that a microRNA is responsible for regulating the level of a predicted E3 ubiquitin ligase in a neural progenitor cell, which affects the viability of its progeny. Identification of the targets of Rchy1 in neural progenitor cells may provide new insights into the control of their differentiation and survival (Foo, 2017a).

Loss of glia in the miR-31a mutant points to a previously unidentified plasticity of the young adult brain in Drosophila. These findings imply ongoing replacement of glia in the young adult brain. Furthermore, they provide evidence that the brain can detect when there are too few glia and that this can trigger progenitor cells to increase production of new glia, by increasing the proportion of their progeny that differentiate into glia. How the number of glia is monitored in the brain will be a topic for future study (Foo, 2017a).

Approximately half of the glia affected in the miR-31a mutant were astrocytes. In this context, parallels to the mammalian brain may be interesting. In mammals, most astrocytes contact a blood vessel. Since astrocytes rely on the vasculature for survival, it is speculated that there is a matching of astrocytes to blood vessels. The fly does not have a closed circulatory system, so there are no blood vessels in the brain. There must be other mechanisms by which astrocyte number is monitored. An obvious possibility is that the need for astrocytes (or their survival) is linked to the number of neurons with which they make contact (Foo, 2017a).

Two recent studies have shown that mammalian glia, specifically oligodendrocyte precursor cells (OPC) and microglia, can repopulate the brain after induced loss of the entire populations of these two cell types. It has been demonstrated that OPC differentiation to oligodendrocytes occurs throughout life. As such, the homeostatic response to maintain OPCs in the brain likely reflects the need to replace the OPCs that have either differentiated or died. Their data also demonstrate that there is a continuous turnover of oligodendrocytes in the adult. Astrogenesis has been observed to occur in the prefrontral cortex of mice in response to voluntary exercise. The current findings provide evidence that this normal turnover of astrocytes also occurs in the Drosophila brain and that specific neural progenitor cells maintain an ongoing homeostatic control of astrocyte numbers in the adult Drosophila brain. It will be interesting to learn whether a comparable progenitor population exists in mammals to support astrocyte turnover (Foo, 2017a).

Multipotent neural stem cells generate glial cells of the central complex through transit amplifying intermediate progenitors in Drosophila brain development

The neural stem cells that give rise to the neural lineages of the brain can generate their progeny directly or through transit amplifying intermediate neural progenitor cells (INPs). The INP-producing neural stem cells in Drosophila are called type II neuroblasts, and their neural progeny innervate the central complex, a prominent integrative brain center. This study used genetic lineage tracing and clonal analysis to show that the INPs of these type II neuroblast lineages give rise to glial cells as well as neurons during postembryonic brain development. These data indicate that two main types of INP lineages are generated, namely mixed neuronal/glial lineages and neuronal lineages. Genetic loss-of-function and gain-of-function experiments show that the gcm gene is necessary and sufficient for gliogenesis in these lineages. The INP-derived glial cells, like the INP-derived neuronal cells, make major contributions to the central complex. In postembryonic development, these INP-derived glial cells surround the entire developing central complex neuropile, and once the major compartments of the central complex are formed, they also delimit each of these compartments. During this process, the number of these glial cells in the central complex is increased markedly through local proliferation based on glial cell mitosis. Taken together, these findings uncover a novel and complex form of neurogliogenesis in Drosophila involving transit amplifying intermediate progenitors. Moreover, they indicate that type II neuroblasts are remarkably multipotent neural stem cells that can generate both the neuronal and the glial progeny that make major contributions to one and the same complex brain structure (Viktorin, 2011).

This study used a combination of genetic lineage tracing, clonal MARCM techniques and molecular labeling to study the developmental mechanisms that give rise to the glial cells of the amplifying type II neuroblast lineages. A novel mode of neurogliogenesis in these lineages was uncovered that involves transit amplifying INPs, which can generate glial cells as well as neurons. This analysis also shows that lineal INP-derived glial and neuronal cells both make major contributions to the central complex. INP-derived neurons project into the neuropile compartments of the central complex and INP-derived glial cells surround and delimit these compartments while undergoing clonal expansion through local proliferation (Viktorin, 2011).

The majority of the glial cells in the adult brain are generated postembryonically. Hitherto unidentified neuroglioblasts have been postulated to give rise to the bulk of these adult-specific glial cells, although some of these arise via proliferative glial cell divisions. This paper reports the identification of type II neuroblasts as neural stem cells with neuroglioblast function in postembryonic development of the central brain. Previous work has shown that these amplifying type II neuroblasts augment proliferation through the generation of INPs resulting in the generation of remarkably large neuronal lineages. These data indicate that type II neuroblasts also generate glial progeny through INPs and, hence, reveal a novel form of CNS neurogliogenesis that involves transit amplifying cells (Viktorin, 2011).

Although type II neuroblasts represent a new type of neuroglioblast in Drosophila brain development, the six identified type II neuroblasts are heterogeneous in terms of their gliogenic activity. While four of these neuroblasts generate comparable numbers of glial cells, a fifth neuroblast (DM4) gives rise to only a few glial cells and the sixth (DM6) rarely generates glia during larval development. This heterogeneity in gliogenic activity is also reflected in the INPs of these type II lineages. Thus, while mixed glial/neuronal INP clones were recovered for most type II lineages, all of the INP clones that contained glia in the DM5 lineage were purely glial, and no glial INP clones were recovered for DM6. Despite this heterogeneity, the generation of INP-derived glial cells in all of these type II lineages appears dependent on the gcm gene. Indeed, this seems to be a feature common to most glial progenitors in the embryonic and postembryonic CNS (Viktorin, 2011).

While glia can derive from the very first larval INPs produced by DM neuroblasts, glia differentiate late in the progression of the lineage, at a time when neuroblast markers are no longer present in the INP. At that time, lineages already contain large amounts of differentiating cells, positive for Prospero and/or the neural differentiation marker Elav. Interestingly, both Prospero and Elav are also found in many young glia, some of them verified to be part of Type II-derived lineages. This co-expression is reminiscent of embryonic glia that have been reported to transiently express Elav, which may thus be a common feature of glial cells also in the larval brain. In addition, there may be Prospero-positive, gliogenic ganglion mother cells that could divide symmetrically to contribute to the variable number of glia observed in late larval INP lineages (Viktorin, 2011).

Neural stem cells in the mammalian brain, notably the radial glia of the cortex, also represent mixed progenitors that can give rise to both neuronal and glial cells in different proliferative modes, and one of these proliferative modes involves transit amplifying INPs. Moreover, some of the amplifying INPs involved in mammalian neocortex development are thought to give rise to neuronal progeny while others give rise to glial progeny. The amplification of proliferation through INPs has been postulated to be fundamental for the increase in cortical size during evolution. The fact that a very comparable mode of INP-dependent proliferation operates in the generation of complex brain architecture in Drosophila suggests that this might represent a general strategy for increased size and complexity in brain development and evolution (Viktorin, 2011).

Previous work has shown that a large subset of the neurons generated by type II neuroblasts contributes to the development of the central complex. The data presented in this study indicate that the INP-derived glial cells from the type II lineages are also involved in central complex development. INP-derived glial cell bodies associate with the larval primordium and, throughout pupal development; they surround and delimit all of the compartments of the differentiating central complex neuropile. Thus neural and glial cells from the same neuroblast lineage participate in the development of the same complex brain neuropile. This reveals a remarkable multipotential nature of the type II neuroblasts; they are neural stem cells that have the potential to generate both neural and glial cells of one and the same complex brain structure (Viktorin, 2011).

Neuroblast lineages have been viewed as 'units of projection' in that the neurons of a given lineage often project their axons along a common trajectory and contribute to the formation of a common neuropile; this is exemplified by the four neuroblast lineages that give rise to the intrinsic cells of the mushroom body neuropile. The neurons of the type II neuroblast lineages do not strictly conform to this notion, since subsets of neurons in the lineage project to different parts of the brain. However, for the large subset of neurons in the type II lineages that project to the developing central complex, the notion of a lineal 'unit of projection' is valid. Indeed this concept can be expanded to include the lineally related glial cells that also contribute to the development of the same neuropile compartment (Viktorin, 2011).

In type II neuroblast clones, most of the INPs, as well as the cell bodies of the neurons that they produce, remain clustered together in the peripheral cell body layer of the brain. Although a few of the INP-derived glial cells are also found in these clusters, most are not. During larval and pupal development, the majority of the INP-derived glial cells are found in or near midline commissural structures such as the central complex precursor (late larva) or the developing central complex neuropile (pupa) (Viktorin, 2011).

One reason for this is that INP-derived glial cells probably migrate away from their site of origin to a different site of final differentiation. Migration of glial cells during CNS development is a common feature in many species and has been studied in detail in the developing ventral nerve cord and optic lobes of Drosophila. Migration of glial cells has also been reported in postembryonic development of the central brain, and in some cases the migrating cells appear to form clusters suggesting that they might derive from common progenitors (Viktorin, 2011).

Another reason for the fact that so many INP-derived glial cells are found in or near the developing central complex is that they proliferate locally. This implies that INP-derived glial cells can undergo clonal expansion in the neuropile. Clonal expansion has been described for the perineurial glial cells localized on the surface of the brain and has also been postulated to take place during the postembryonic development of neuropile glial cells. Since INP-derived glial cells undergo substantial (at least fourfold) proliferative clonal expansion, it will be important to determine what controls their mitotic activity. In view of the vulnerability of the amplifying type II neuroblast lineages to overproliferation and brain tumor formation, a tight control of self-renewing glial cell proliferation in these lineages is likely to be essential (Viktorin, 2011).

Organization and function of the blood-brain barrier in Drosophila

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. This study presents 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, 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, were characterized. To test the functional roles of these sheaths a series of dye penetration experiments were performed in the nervous systems of wild-type and mutant embryos. Comparing the kinetics of uptake of different sized fluorescently labeled dyes in different mutants led to the conclusion 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). The requirements of different septate junction components were compared for the integrity of the blood-brain barrier, and evidence is provided that two of the six Claudin-like proteins found in Drosophila are needed for normal blood-brain barrier function (Stork, 2008).

Fast neuronal conductance requires a tight electrical insulation of the axons and in the mammalian nervous system, myelin and saltatory conductance evolved. Arthropods have not evolved saltatory conductance, but they are nevertheless in need for fast electrical conductance. In this respect it is not surprising that in marine shrimps myelin-like structures have been described . Drosophila follows two different and seemingly independent strategies to ensure fast conductance. In some central neuronal networks large caliber axons develop, whereas in the peripheral nervous system axons are insulated by several glial sheaths to ensure insulation. Initially, at the beginning of larval life, the different sensory and motor axons are kept as separate fascicles within the segmental nerves, suggesting there might be some degree of electrical cross talk within the different modalities. As the larva matures, the inner wrapping glia starts to grow around single axons, which may allow more sophisticated movements of the wandering larvae (Stork, 2008).

Whereas the wrapping glia insulates individual axons, do perineurial and subperineurial glia insulate the entire nervous system and set up the blood-brain-barrier? Genetic experiments and ultrastructural studies have long indicated that septate junctions provide the most effective part of this barrier. Indeed, the subperineurial cell are formed early in development and these cells are connected by septate junctions from late embryonic stages onwards. Using Gal4 driver strains specific to the subperineurial cells as well as in vivo septate junction markers, this study confirms that during larval life the subperineurial cells do not divide but grow enormously large in size. Septate junctions formed by the subperineurial cells are mostly found in interdigitated zones of cell-cell contact. Cell division would likely require disintegration of septate junctions and thus result in a temporal opening of the blood-brain barrier, which would be deleterious for the animal. This is in agreement with previous findings that Gliotactin expressing cells, forming septate junctions, do not divide during larval live (Stork, 2008).

The outermost glial cell layer is formed by the perineurial cells. Although these cells have long been described, their origin is still a matter of debate. In EM micrographs of late embryonic staged peripheral nerves some perineurial cells can be detected apically to the subperineurial cells. These glial cells divide during larval life and generate a large number of fine cell protrusions that cover the subperineurial cells. One function of the perineurium might be to influence the development and/or the tightness of the subperineurial layer. A comparable cellular function has been attributed to the astrocytes in the mammalian nervous system. Alternatively, the perineurial glial cells might provide a cellular basis for the response to injury. Unfortunately, to date there is no specific driver strains that allow manipulation of this glial cell population. Interestingly, a reverse relationship between subperineurial and perineurial cells has been suggested previously as subperineurial expression of activated Ras or PI3K (phosphoinositide 3-kinase) resulted in an thickening of the perineurial sheath (Stork, 2008).

Additionally, the fray gene has been shown to be required for normal axonal ensheathment. Interestingly the mutant phenotype could be rescued by expressing fray using three different Gal4 drivers. After the analysis of the specificity of these drivers (Mz317, subperineurial glia and weak wrapping glia; Mz709, all glial cell types; gliotactinGal4, subperineurial glia), it is concluded that fray is expressed in subperineurial glia and controls axonal ensheathment of wrapping glia in a noncell autonomous manner (Stork, 2008).

Given the different cellular barriers described in this report, questions arise concerning the functional contributions of the different layers. Kinetic studies were performed that supported the importance of the septate junctions in particular for small components. Animals lacking septate junctions are as leaky to a 10 kDa dextran as animals lacking all glial cell layers. However, when it comes to larger molecules, the relevance of the other cell layers becomes obvious. Although a 500 kDa dextran can easily penetrate into the nervous system of a glial cells missing embryo, its leakage into the nervous system of a neurexinIV mutant lacking septate junctions is greatly reduced. Thus, the other layers contribute to the function of the blood-brain barrier. Because a continuous perineurium is not fully formed in first instar larvae, the barrier function has to be assigned to the neural lamella and the inner glial layer. There are several reports showing that the neural lamella can act as an efficient filter for heavy metal ions. Possibly, large molecules such as the 500 kDa dextran are also trapped in this ECM. Alternatively, large particles are stopped by the diffusion barrier established by the normal cell-cell contacts between subperineurial cells and inner glial cell types like wrapping glia in the peripheral nerves and cortex and neuropile glia in the CNS (Stork, 2008).

The diffusion barrier provided by glial cells or epithelial sheaths is generated by special junctional complexes that help to tightly associate the involved cells. Drosophila epithelia as well as glial cells are characterized by septate junctions. Quite similar structures are also found at the mammalian paranodal junctions, which provide the structural basis for the tight electrical insulation of the nerve. A core component of the mammalian axoglial septate junctions is the NeurexinIV homolog Caspr that together with its binding partners, Contactin and Neurofascin155, sets up a tripartite adhesion complex at the paranode (Stork, 2008).

The function of this complex appears conserved in Drosophila, although there are some notable differences. The Caspr homolog NeurexinIV is expressed by glial cells as are Contactin and the Neurofascin155 homolog Neuroglian. As a consequence, in the fly septate junctions are formed between glial cells, whereas they are formed between neuronal and glial membranes in the mammalian system. The Caspr/Contactin/Neurofascin155 complex seals the paranodal junction and a similar function has been attributed to this protein complex in the invertebrate blood-brain barrier. This study found a less pronounced function of Contactin compared with NeurexinIV for the blood-brain barrier establishment, corroborating findings made in embryonic epithelia (Stork, 2008).

Another prominent component of the junctional complexes are the Claudin proteins. In mammals, members of these four transmembrane domain proteins are associated with tight junctions that are often considered to be functionally equivalent to the invertebrate septate junctions. In Drosophila two Claudin-like proteins have been described to be required for formation of normal epithelial barrier formation. This study shows that both Sinuous and Megatrachea are also needed for the establishment of normal blood-brain barrier formation. Similarly, it was shown that mammalian claudin5 is a major component of tight junctions of brain endothelial cells. claudin5 mutant mice show no structural or ultrastructural deficits, but have an impaired blood-brain barrier. The association of Claudins integrated in opposing membranes is thought to provide pores that can control the paracellular diffusion of small molecules. Although Drosophila Sinuous and Megatrachea clearly contribute to the barrier function, it is inconceivable that fly Claudins traverse the 20 nm wide septate gap to form a Claudin pore as it is discussed for the vertebrate Claudins. It has also been suggested that invertebrate Claudins might have lost their pore-like functions and exert only signaling function to establish the barrier (Stork, 2008).

Such a signaling function may control the size selectivity of the barrier and indeed sinuous mutants show only a weak barrier phenotype comparable with moody mutants, correlating with reduced septate junctions. This study demonstrates that a loss of septate junctions associated with neurexinIV mutants results in breakdown of the blood-brain barrier comparable with what is observed in animals lacking all glial cells. However, additional mechanisms are in place to control the paracellular diffusion of larger particles. A 500 kDa dextran can easily penetrate the nervous system of a glial cells missing embryo but cannot enter a nerve cord only lacking septate junctions (Stork, 2008).

Analysis of glial distribution in Drosophila adult brains

Neurons and glia are the two major cell types in the nervous system and work closely with each other to program neuronal interplay. Traditionally, neurons are thought to be the major cells that actively regulate processes like synapse formation, plasticity, and behavioral output. Glia, on the other hand, serve a more supporting role. To date, accumulating evidence has suggested that glia are active participants in virtually every aspect of neuronal function. Despite this, fundamental features of how glia interact with neurons, and their spatial relationships, remain elusive. This study describes the glial cell population in Drosophila adult brains. Glial cells extend and tightly associate their processes with major structures such as the mushroom body (MB), ellipsoid body (EB), and antennal lobe (AL) in the brain. Glial cells are distributed in a more concentrated manner in the MB. Furthermore, subsets of glia exhibit distinctive association patterns around different neuronal structures. Whereas processes extended by astrocyte-like glia and ensheathing glia wrap around the MB and infiltrate into the EB and AL, cortex glia stay where cell bodies of neurons are and remain outside of the synaptic regions structured by EB or AL (Ou, 2016).

Drosophila cortex and neuropile glia influence secondary axon tract growth, pathfinding, and fasciculation in the developing larval brain

Glial cells play important roles in the developing brain during axon fasciculation, growth cone guidance, and neuron survival. In the Drosophila brain, three main classes of glia have been identified including surface, cortex, and neuropile glia. While surface glia ensheaths the brain and is involved in the formation of the blood-brain-barrier and the control of neuroblast proliferation, the range of functions for cortex and neuropile glia is less well understood. This study used the nirvana2-GAL4 driver to visualize the association of cortex and neuropile glia with axon tracts formed by different brain lineages and to selectively eliminate these glial populations via induced apoptosis. The larval central brain consists of approximately 100 lineages. Each lineage forms a cohesive axon bundle, the secondary axon tract (SAT). While entering and traversing the brain neuropile, SATs interact in a characteristic way with glial cells. Some SATs are completely invested with glial processes; others show no particular association with glia, and most fall somewhere in between these extremes. The results demonstrate that the elimination of glia results in abnormalities in SAT fasciculation and trajectory. The most prevalent phenotype is truncation or misguidance of axon tracts, or abnormal fasciculation of tracts that normally form separate pathways. Importantly, the degree of glial association with a given lineage is positively correlated with the severity of the phenotype resulting from glial ablation. Previous studies have focused on the embryonic nerve cord or adult-specific compartments to establish the role of glia. This study provides, for the first time, an analysis of glial function in the brain during axon formation and growth in larval development (Spindler, 2009).

Secondary neurons, which are born during the larval period, form SATs that have to extend over relatively long distances, finding their way amidst a complex array of (primary) axons, dendrites, and glia. The association of glia and SATs varies for different lineages. SATs either (1) remained wrapped within the neuropile or joined other tracts that were then ensheathed as a larger tract system, (2) encountered strands of glial condensations, or (3) had no association with glial sheaths. The association between individual SATs and glia was highly invariant. Thus, if SAT A joined SAT B to form a larger tract system that became wrapped by glia, the same densities of glia could be observed in other brains for the corresponding SATs (Spindler, 2009).

To address the role of glia during SAT fasciculation, growth, and guidance, cortex and neuropile glia, the two glial types in contact with growing SATs, were selectively eliminate. Expression of the pro-apoptotic proteins Hid and Rpr were effective in inducing apoptosis of most cortex and neuropile glia by the early or mid larval stage. It can be assumed that the primary axon tract formation is not disrupted, given that expression of the nrv2-GAL4 driver line does not set in prior to stage 12, primary axon tract (PAT) patterning is complete before glia invade the neuropile, and the first signs of apoptosis appear after hatching. In addition, the surface glia remain intact, providing general ensheathment around the brain, a functional blood–brain barrier, and potential signaling molecules used to control neuroblast proliferation (Spindler, 2009).

Upon the elimination of glia, frequent abnormalities are seen in the pattern of SATs. Importantly, the strength of an SAT phenotype appears to correlate with the degree of glial association of that SAT in a wild-type brain. Of all SATs analyzed, the mushroom body (associated with a complete glial sheath) exhibits the most severe defects, including complete SAT misguidance and aberrant fasciculation of neurites from adjacent SATs. In contrast, SATs that were less endowed with glia in the wild type typically had a normal projection pattern (Spindler, 2009).

This study does not differentiate between a role for glia in producing chemo-attractant signals or acting as a physical scaffold for guidance. In previous studies, ectopic expression of dominant-negative Drosophila E-cadherin in either cortex/neuropile glia or SATs themselves results in non-radial trajectories of SATs into the neuropile. This suggests a requirement for SAT-glia adhesion as secondary axons project toward the cortex-neuropile boundary. However, the direction of neuroblast division is also disrupted with DE-cadherin knock-down, thus aberrant SAT trajectories may be a secondary effect of abnormal cell body layering within the cortex. In support of a signaling role for glia, the midline guidance defect of the CP1 SAT (anteromedially, crossing the peduncle and entering the diagonal commissure) is reminiscent of the robo-slit phenotypes in the Drosophila ventral nerve cord. Whether robo/slit signaling is also used between SATs and glia in the brain is a question that warrants further investigation (Spindler, 2009).

The situation that SATs in part require glia for pathway guidance in the neuropile is different than the embryonic brain in which pioneer axons and PATs are formed in the absence of extensive glial processes. Why are SATs different? One can imagine the scenario in which the PATs generate the neuropile de novo, forcing cell body movement outwards as the central neuropile grows. By first instar, a full neuropile is established, and SATs must guide through a dense maze of neurites, glia, and trachea. It therefore appears that: (1) PATs establish the initial connectivity of the brain; (2) glia grow in around this initial scaffolding, and finally (3) SATs use both the PAT scaffolding and the glial boundaries for guidance into and around the neuropile (Spindler, 2009).

Insects have long been used to evaluate glial-neuronal interactions from embryonic to adult stages. An important focus of these studies was whether or not glia or axon tracts appear first, and in how far axonal pathfinding is disrupted if glia is ablated. In this regard, clear developmental differences have been found between distinct regions of the nervous system, as well as between insect species for homologous nervous system domains (Spindler, 2009).

In the Drosophila ventral nerve cord, two subpopulations of neuropile glia were studied in the context of axonal pathfinding: midline glia and longitudinal glia. Both types of glial cells appear around the same stage when pioneer neurons extend their axons. The proper number and positioning of midline glia is clearly required for the formation of commissural axon tracts. The loss of longitudinal glia (by ablation and in embryos mutant for the gcm gene) primarily affects the defasciculation and fasciculation events of longitudinal pioneer tracts, subsequently affecting the follower neuron trajectories. Note that gcm is required for all classes of glia, including surface glia; thus, in very late gcm mutant embryos, severe disruptions of the entire neuropile result from the fact that, with the onset of embryonic movement, the CNS lacking surface glia is literally 'shredded to pieces' (Spindler, 2009).

In the embryonic Drosophila peripheral nervous system, ablation of the peripheral glia (i.e. exit glia) via targeted overexpression of the cell death genes grim and ced-3 lead to aberrant pathfinding of both motor and sensory axons as they exited the CNS; although the motor neurons eventually overcame the absence of peripheral glia finding correct muscle targets, suggesting a limited role for the peripheral glia in the initial trajectory of motor neurons. In grasshopper, ablation of the cell-segment boundary glial guidepost cell lead to more severely aberrant axon trajectories. Ablation of glia surrounding the antennal lobe of adult Manduca generates olfactory axon de-fasciculation and misguidance. Finally, guidance phenotypes have also been observed with disruption of the Drosophila lamina glia; R1-R6 photoreceptor axons show aberrant guidance past the lamina into the medulla of the optic lobe (Spindler, 2009 and references therein).

An important aspect of the developmental role of glia added by this study is the focus on the correlation between closeness of axon tract/glia association, and axon tract abnormality in the absence of glia. In other words: the nervous system is formed by a large number of fascicles, and these fascicles vary in their degree of glial wrapping. It would be misleading when carrying out a genetic study to only focus on a single fascicle (or small subset of fascicles), and extrapolate from the phenotype observed for this fascicle onto the brain as a whole. In this analysis, SATs of several lineages, notably those that in normal brains have little glia covering them in the neuropile, show few abnormalities in glia-less brains. By contrast, other lineages were affected in the majority of cases, and typically, these lineages also were the ones whose SATs were associated more closely with glia (Spindler, 2009).

Neuropile compartments are formed by terminal branches of axons and dendrites and their synapses. For example, the antennal lobe of insects consists of the axonal terminals of sensory neurons located in the antenna, and dendritic terminals of antennal projection neurons and local interneurons located in the deuterocerebrum, aside from a relatively small number of other modulatory neurons. Sensory neurons expressing the same olfactory receptor all converge onto the dendrites of a small number of projection neurons to form an olfactory glomerulus. In many insects, notably Manduca, olfactory glomeruli are individually compartmentalized by neuropile glia, and it has been shown that glia plays a prominent role in establishing the glomeruli organization. According to the prevailing view, glomeruli are initially ordered by the specialized endings of sensory terminals into protoglomeruli; however, glial processes soon invade the space in between protoglomeruli and restrict the arborization of receptor axons. Therefore, in Manduca antennal lobes, glia is required for early maintenance of the glomerular map (Spindler, 2009 and references therein).

A recent analysis of the time course of glial development in the Drosophila antennal lobe suggested that in this species, glia plays an even lesser role, since glomeruli are only incompletely, and at a late time point, wrapped by glia. While glia were never ablated in those studies, later work found that elimination of Neuroglian from midline glia resulted in an inability of ORN axons to cross through the antennal commissural tract to the contralateral lobe. In this study, most adult brains lacking cortex and neuropile glia still form antennal lobes with glomeruli, however in some samples the glomeruli are poorly defined, and cannot be identified by their position and shape in the antennal lobe. The variability in phenotype penetrance likely stems from larvae containing the most glia surviving to eclosion; therefore, a relatively normal looking adult brain could stem from a weak glial apoptosis early in development. Whether the antennal lobe disorganization is a secondary defect due to a lack of definition normally provided by the presence of glia, a result of SATs that normally contribute to the AL misguiding or truncating early, or a maintenance defect in which axons begin inappropriately intermingling among glomeruli is not clear. Perhaps the Drosophila antennal lobe glia is required for glomeruli maintenance even after glomeruli organization is established, and future studies will hopefully address this possibility (Spindler, 2009).

In the post-embryonic midline of Drosophila, ablation of the transient interhemispheric fibrous ring (TIFR), a transient population of midline glia, by ectopic expression of the pro-death gene hid, generates defects in the adult central complex. What is unclear, however, is the cellular event that is effected to cause the defects. This study suggests that morphological abnormalities in adult compartments from glial manipulation are due to the misguidance of larval SATs to the correct neuropile compartment in the brain, affecting the formation of adult neuropile structures. This study presents evidence that glia is an important mediator of axon guidance in the Drosophila larval brain; the mechanism for glia-neuron communication during this process is an exciting area for future investigation (Spindler, 2009).

Polyploidization of glia in neural development links tissue growth to blood-brain barrier integrity

Proper development requires coordination in growth of the cell types composing an organ. Many plant and animal cells are polyploid, but how these polyploid tissues contribute to organ growth is not well understood. This study found the Drosophila subperineurial glia (SPG) to be polyploid, and ploidy is coordinated with brain mass. Inhibition of SPG polyploidy caused rupture of the septate junctions necessary for the blood-brain barrier. Thus, the increased SPG cell size resulting from polyploidization is required to maintain the SPG envelope surrounding the growing brain. Polyploidization likely is a conserved strategy to coordinate tissue growth during organogenesis, with potential vertebrate examples (Unhavaithaya, 2012).

How different tissues coordinate their growth to form the final, properly sized organ is not well understood. SPG envelop neurons via septate junctions to create the blood-brain barrier during Drosophila development. This strategy creates a developmental conundrum, because neurons continue to divide and increase in mass, while SPG need to both maintain a tight seal and increase cell size to accommodate neuronal growth. This study has demonstrate that by increasing cell size via increases in ploidy, SPG can keep the cell number constant and maintain the septate junctions unperturbed by cytokinesis. The experiments suggest that there exists feedback between the polyploidizing SPG tissue and the mitotic neuronal tissue to coordinate SPG ploidy with brain lobe size. Consistent with this model, the SPG of the brain lobes, which must cover a larger surface area than those in the ventral cord, attain a higher level of ploidy and cell size. It remains to be determined why some SPG are multinucleated and others have a single polyploid nucleus and why this is restricted to SPG in the brain. This study presents a novel mechanism for how different tissues are scaled to size in a developing brain, as well as establishing a new paradigm for the use of polyploidization to coordinate growth of different tissues during organogenesis (Unhavaithaya, 2012).

The strategy of using polyploidization to coordinate tissue growth appears to be conserved in vertebrates and to function in other organ systems. Examples are the polyploidizing trophoblasts that surround the mitotic blastula during embryo implantation, as well as the polyploid keratinocytes situated atop the dividing basal cell layer in the skin. Both trophoblasts and keratinocytes form tight junctions. In trophoblasts, these are proposed to shield embryos from the potential harmful factors in the maternal environment, whereas in keratinocytes, the tight junctions provide a barrier for the skin epithelia. Polyploidy is thus a likely strategy by which these polyploid tissues achieve growth while maintaining tight junctions. The signaling pathways these tissues and the SPG use for growth coordination remain to be defined. The coordination of SPG and brain growth in Drosophila reveals a mechanism of organogenesis that may have evolved to act also in vertebrate tissues with polyploid cells to control proper development of the tissue layers (Unhavaithaya, 2012).

Regulation of Drosophila glial cell proliferation by Merlin-Hippo signaling

Glia perform diverse and essential roles in the nervous system, but the mechanisms that regulate glial cell numbers are not well understood. This study identified and characterized a requirement for the Hippo pathway and its transcriptional co-activator Yorkie in controlling Drosophila retinal and central brain glia proliferation. Yorkie is both necessary for normal glial cell numbers and, when activated, sufficient to drive glial over-proliferation. Yorkie activity in glial cells is controlled by a Merlin-Hippo signaling pathway, whereas the upstream Hippo pathway regulators Fat, Expanded, Crumbs and Lethal giant larvae have no detectable role. Functional characterization of Merlin-Hippo signaling was extended by showing that Merlin and Hippo can be physically linked by the Salvador tumor suppressor. Yorkie promotes expression of the microRNA gene bantam in glia, and bantam promotes expression of Myc, which is required for Yorkie and bantam-induced glial proliferation. These results provide new insights into the control of glial growth, and establish glia as a model for Merlin-specific Hippo signaling. Moreover, as several of the genes studied have been linked to human gliomas, the results suggest that this linkage could reflect their organization into a conserved pathway for the control of glial cell proliferation (Reddy, 2011).

Merlin was first identified as the product of a human tumor suppressor gene, NF2, loss of which in peripheral glial cells results in benign tumors. Merlin has also been identified as an inhibitor of gliomas. Observations in this study indicate that the role of Merlin as a negative regulator of glial cell proliferation is conserved from humans to Drosophila and, thus, that Drosophila can serve as a model for understanding Merlin-dependent regulation of glial growth (Reddy, 2011).

Studies in Drosophila imaginal discs first linked Merlin to Hippo signaling, and Merlin was subsequently linked to Hippo signaling in mammalian cells, including its role in meningioma. However, the tumor suppressor activity of Merlin has also been linked to other downstream effectors in mammals, including Erb2, Src, ras, rac, TORC1 (CRTC1 - Human Gene Nomenclature Database; see Drosophila CRTC) and CRL4 (IL17RB - Human Gene Nomenclature Database), creating some uncertainty regarding the general importance of the linkage of Merlin to Hippo in growth control. It was found that depletion of Merlin, depletion of other tumor suppressors in the Hippo pathway, or expression of an activated form of Yki, all result in similar glial overgrowth phenotypes. Moreover, depletion of Merlin increased nuclear localization of Yki, and depletion of Yki suppressed the overgrowth phenotype of Merlin. Together, these observations clearly establish that the glial overgrowth phenotype associated with Merlin depletion in Drosophila is mediated through the Hippo signaling pathway (Reddy, 2011).

A noteworthy feature of Hippo signaling in Drosophila glial cells is that Merlin appears to be uniquely required as an upstream regulator of Hippo signaling, as the Fat-dependent, Ex-dependent and Lgl-dependent branches have no detectable role. Glia might, thus, provide an ideal model for mechanistic investigations of the Merlin branch of Hippo signaling. Fat-Hippo signaling employs Fat as a transmembrane receptor and Dachsous as its transmembrane ligand, whereas Ex-Hippo signaling appears to employ Crumbs as a transmembrane receptor and ligand. By contrast, Drosophila transmembrane proteins that mediate extracellular signaling and interact with Merlin have not yet been identified. Distinct mechanisms might also be involved in signal transduction downstream of Merlin. Although there is evidence that Ex and Merlin can both influence Hippo activity, Ex, but not Mer, can directly associate with Hpo. Conversely, Merlin, but not Ex, can interact directly with Salvador, and Merlin, Salvador and Hippo can form a trimeric complex. Moreover, the kibra loss-of-function phenotype is weaker than expanded in imaginal discs, but comparable to Merlin, and it was found that depletion of kibra also has a significant effect on glial cell proliferation. Kibra is highly expressed in mammalian brain, and alleles of KIBRA (WWC1 - Human Gene Nomenclature Database) have been linked to human memory performance. The role of kibra in regulating glial cell numbers in Drosophila thus raise the possibility that the influence of KIBRA on human memory might reflect a role in glial cells (Reddy, 2011).

Finally, it is noted that although Hippo signaling has been investigated in several different organs in Drosophila, including imaginal discs, ovarian follicle cells, neuroepithelial cells and intestinal cells, these all involve roles in epithelial cells, in which upstream regulators of the pathway (e.g., Fat, Ex, Mer) all have a distinctive localization near adherens junctions. The identification of a requirement for Hippo signaling in glia is the first time in Drosophila that a role for the pathway has been identified in non-epithelial cells. Indeed, in previous studies it was found that Hippo signaling influences proliferation of neuroepithelial cells, but other neuronal cell types, including neuroblasts, ganglion mother cells and neurons, are insensitive to Yki (Reddy, 2011).

Considerable attention has been paid to genes for which mutation or inappropriate activation can cause over-proliferation of glial cells, resulting in glial tumors. However, less is known about the mechanisms required for normal glial growth. Through loss-of-function studies, several genes were identified that are essential for normal glial cell numbers, including yki, sd, ban, mad and myc. The requirement for yki, mad and sd, together with epistasis studies, identifies a requirement for active Yki in glial growth. This in turn implies that downregulation of Hippo signaling is important for normal glial growth. Understanding how this is achieved will provide further insights into the regulation of glial cell numbers (Reddy, 2011).

A requirement for Mad, together with its upstream regulator Thickveins (Tkv), in promoting retinal glial cell proliferation has been described previously. The current studies of glial cells, together with recent work in imaginal discs, emphasize that in mediating the growth-regulating activity of Hippo signaling, Yki utilizes multiple DNA-binding partners (i.e. Mad and Sd) in the same cells at the same time to regulate distinct downstream target genes required for tissue growth (Reddy, 2011).

Although Yki activity influenced glial cell numbers throughout the nervous system, direct analysis of cell proliferation by Ethynyl deoxyuridine (EdU) labeling revealed that retinal glia were more sensitive to Yki activation at late third instar than central brain glia, and significant induction of central brain glial cell proliferation was only observed when Yki activation was combined with Myc over-expression. Further studies will be required to define the basis for this differential sensitivity, but the implication that the proliferative response to Yki is modulated by developmental stage and/or glial cell type has important implications for diseases associated with both excess and deficits of glial cells (Reddy, 2011).

These studies in Drosophila delineate functional relationships among genes involved in the control of glial cell proliferation. Mammalian homologs of Merlin, Yki and Myc have been implicated in glioma. Although a mammalian homolog of ban has not been described, other miRNAs have also been linked to glioma. The current observations imply that these genes can be placed into a pathway, in which Merlin, through Hippo signaling, regulates Yki, Yki regulates ban, and ban regulates Myc. However, as expression of Myc alone did not lead to substantial overgrowth of glia, Yki and ban must also have other downstream targets important for the promotion of glial cell proliferation. Moreover, the observations indicate that a Yki-Sd complex is also required for glial growth. In addition to the well characterized downstream target Diap1, Yki-Sd complexes in glial cells might regulate Myc directly, as suggested by studies in imaginal disc, and might regulate cell cycle genes in conjunction with E2F1 (Reddy, 2011).

The influence of activated-Yki on a ban-GFP sensor, together with the observations that yki is not required for ban-mediated overgrowth, whereas ban is required for Yki-mediated overgrowth, position ban downstream of Yki. This is consistent with studies of Hippo signaling in imaginal discs, in which ban has also been identified as a target of Yki for growth regulation. The placement of Myc downstream of Yki and ban is supported by the observation that Myc levels can be increased by expression of ban or activated-Yki, and by genetic tests that indicate that Myc is required for Yki- and ban-promoted glial overgrowth. A mechanism by which ban can regulate Myc levels, involving downregulation of a ubiquitin ligase that negatively regulates Myc, was identified recently in imaginal discs, and might also function in glial cells. Myc has been reported to downregulate Yki expression in imaginal discs and, although whether a similar negative-feedback loop exists in glial cells has not been investigated, the synergistic enhancement of glial cell proliferation observed when Yki and Myc were co-expressed is consistent with this possibility, as the expression of both genes under heterologous promoters could bypass negative regulation of Yki by Myc (Reddy, 2011).

The Myc proto-oncogene is de-regulated or amplified in several human cancers, including gliomas. The sensitivity of Yki/ban-induced overgrowth to reduced Myc levels parallels studies of glioma models involving other signaling pathways. For example, Myc is upregulated by EGFR, and is limiting for EGFR-PI3K-induced glial cell overgrowth in a Drosophila glioma model, and p53 and Pten-driven glioma in mouse models is also Myc dependent. Considering the evidence linking Merlin and Yap to glial growth in mammals, and the identification of Myc as a downstream target of Yap in cultured cells, it is likely that Yap could also influence glial growth in mammals, in part, through regulation of Myc (Reddy, 2011).

Concerted control of gliogenesis by InR/TOR and FGF signalling in the Drosophila post-embryonic brain

Glial cells are essential for the development and function of the nervous system. In the mammalian brain, vast numbers of glia of several different functional types are generated during late embryonic and early fetal development. However, the molecular cues that instruct gliogenesis and determine glial cell type are poorly understood. During post-embryonic development, the number of glia in the Drosophila larval brain increases dramatically, potentially providing a powerful model for understanding gliogenesis. Using glial-specific clonal analysis this study found that perineural glia and cortex glia proliferate extensively through symmetric cell division in the post-embryonic brain. Using pan-glial inhibition and loss-of-function clonal analysis it was found that Insulin-like receptor (InR)/Target of rapamycin (TOR) signalling is required for the proliferation of perineural glia. Fibroblast growth factor (FGF) signalling is also required for perineural glia proliferation and acts synergistically with the InR/TOR pathway. Cortex glia require InR in part, but not downstream components of the TOR pathway, for proliferation. Moreover, cortex glia absolutely require FGF signalling, such that inhibition of the FGF pathway almost completely blocks the generation of cortex glia. Neuronal expression of the FGF receptor ligand Pyramus is also required for the generation of cortex glia, suggesting a mechanism whereby neuronal FGF expression coordinates neurogenesis and cortex gliogenesis. In summary, this study has identified two major pathways that control perineural and cortex gliogenesis in the post-embryonic brain and has shown that the molecular circuitry required is lineage specific (Avet-Rochex, 2012).

The correct control of gliogenesis is crucial to CNS development and the Drosophila post-embryonic nervous system is a powerful model for elucidating the molecular players that control this process. This study has identified two separate glial populations that proliferate extensively and have defined the key molecular players that control their genesis and proliferation. Perineural and cortex glia both use insulin and FGF signalling in a concerted manner, but the requirements for these pathways are different in each glial type. The data suggest a model that describes the molecular requirements for post-embryonic gliogenesis in each of these glial types in the brain (Avet-Rochex, 2012).

The results show that Pyramus is expressed by perineural glia to activate FGF signalling in adjacent glia and acts in parallel to InR/TOR signalling (activated by the expression of Dilp6). These two pathways act synergistically to generate the correct complement of perineural glia. The results also show that cortex glia proliferation is controlled by FGF signalling through FGFR (Htl) and the Ras/MAPK pathway. Pyr expression is required from both glia and neurons and acts non-cell-autonomously. Neuronal Pyr expression activates the FGFR on adjacent cortex glia, thereby coordinating neurogenesis and glial proliferation. InR is also partially required in cortex glia and is likely to signal through the Ras/MAPK pathway (Avet-Rochex, 2012).

Using both pan-glial inhibition and LOF clonal analysis this study has shown that the InR/TOR pathway is required for perineural glia proliferation. InR/TOR signalling has widespread roles in nervous system development and a role has been demonstrated for this pathway in the temporal control of neurogenesis (Bateman, 2004; McNeill, 2008). InR can be activated by any one of seven DILPs encoded by the Drosophila genome, which can act redundantly by compensating for each other. dilp6 is expressed in most glia during larval development, including perineural and cortex glia, and that dilp6 mutants have reduced gliogenesis. The dilp6 phenotype is weaker than that associated with the inhibition of downstream components of the InR/TOR pathway, suggesting that other DILPs might be able to compensate for the absence of dilp6 expression in glia (Gronke, 2010). Pan-glial inhibition and clonal analysis also demonstrated that the FGF pathway is required for normal levels of perineural glia proliferation. FGF signalling is activated in perineural glia by paracrine expression of Pyr. Inhibition of either the InR/TOR or FGF pathway reduced perineural glia proliferation by about half, so tests were performed to see whether these two pathways act together. The data demonstrate that inhibition of both pathways simultaneously has a synergistic effect, suggesting that these two pathways act in parallel, rather than sequentially, and that their combined activities generate the large numbers of perineural glia found in the adult brain (Avet-Rochex, 2012).

Cortex glia employ a molecular mechanism distinct from that of perineural glia to regulate their proliferation. Cortex glia have a clear requirement for InR, as InR mutant cortex clones are significantly reduced in size. The early events in post-embryonic gliogenesis are poorly understood, but FGF signalling is likely to be required during this stage as LOF clones for components of this pathway almost completely block cortex gliogenesis. These data suggest that InR acts in parallel to FGF signalling in these cells, as loss of InR combined with activation of FGF signalling only partially rescues the InR phenotype. Interestingly, the PI3K/TOR pathway is not required in cortex glia, suggesting that InR signals through the Ras/MAPK pathway to control cortex glia proliferation (Avet-Rochex, 2012).

The FGF pathway in cortex glia responds to paracrine Pyr expression from both glia and neurons. Expression from both glia and neurons is required to activate the pathway and stimulate cortex gliogenesis. Neuronal regulation of glial FGF signalling enables cortical neurogenesis to modulate the rate of gliogenesis, so that the requisite number of glia are generated to correctly enwrap and support developing cortical neurons. Recent studies have also identified a mechanism by which DILP secretion by glia controls neuroblast cell-cycle re-entry in the Drosophila early post-embryonic CNS. Thus, neurons and glia mutually regulate each other's proliferation to coordinate correct brain development (Avet-Rochex, 2012).

This study has shown that two major glial populations in the larval brain, perineural and cortex glia, are generated by glial proliferation rather than differentiation from neuroglioblast or glioblast precursors. Differentiation of most embryonic glia from neuroglioblasts in the VNC requires the transcription factor glial cells missing (gcm), which is both necessary and sufficient for glial cell fate. In the larval brain the role of gcm is much more restricted and it is not expressed in, nor required for, generation of perineural glia. Thus, the developmental constraints on gliogenesis in the embryonic and larval CNS are distinct. The larval brain undergoes a dramatic increase in size during the third instar, which might favour a proliferative mode, rather than continuous differentiation from a progenitor cell type (Avet-Rochex, 2012).

Glial dysfunction is a major contributor to human disease. The release of toxic factors from astrocytes has been suggested to be a contributory factor in amyotrophic lateral sclerosis and astrocytes might also play a role in the clearance of toxic Aβ in Alzheimer's disease. Rett syndrome is an autism spectrum disorder caused by LOF of the transcription factor methyl-CpG-binding protein 2 (MeCP2). Astrocytes from MeCP2-deficient mice proliferate slowly and have been suggested to cause aberrant neuronal development. This hypothesis was recently confirmed by astrocyte-specific re-expression of Mecp2 in MeCP2-deficient mice, which improved the neuronal morphology, lifespan and behavioural phenotypes associated with Rett syndrome. Characterisation of the molecular control of gliogenesis during development might lead to a better understanding of such diseases (Avet-Rochex, 2012).

Antagonistic feedback loops involving rau and sprouty in the Drosophila eye control neuronal and glial differentiation

During development, differentiation is often initiated by the activation of different receptor tyrosine kinases (RTKs), which results in the tightly regulated activation of cytoplasmic signaling cascades. In the differentiation of neurons and glia in the developing Drosophila eye, this study found that the proper intensity of RTK signaling downstream of fibroblast growth factor receptor (FGFR) or epidermal growth factor receptor require two mutually antagonistic feedback loops. A positive feedback loop was identified mediated by the Ras association (RA) domain-containing protein Rau (CG8965) that sustains Ras activity and counteracts the negative feedback loop mediated by Sprouty. Rau has two RA domains that together show a binding preference for GTP (guanosine 5'-triphosphate)-loaded (active) Ras. Rau homodimerizes and is found in large-molecular weight complexes. Deletion of rau in flies decreases the differentiation of retinal wrapping glia and induces a rough eye phenotype, similar to that seen in alterations of Ras signaling. Further, the expression of sprouty is repressed and that of rau is increased by the COUP transcription factor Seven-up in the presence of weak, but not constitutive, activation of FGFR. Together, these findings reveal another regulatory mechanism that controls the intensity of RTK signaling in the developing neural network in the Drosophila eye (Sieglitz, 2013).

During development, often single bursts of RTK activity suffice to direct important cellular decisions. In other cases, multiple rounds of RTK activation are required to trigger a certain reaction profile, and in yet other cases, such as in the developing eye imaginal disc, a sustained low level of EGFR activity is needed. This study identified the Drosophila RA domain containing protein Rau, which constitutes the first cell-autonomous positive feedback regulator acting on both EGFR- and FGFR-induced signaling. In the developing fly compound eye, it was found that sustained RTK activity is modulated through a positive feedback loop initiated by Rau, which is counterbalanced by the negative regulator Sprouty. The balance of these two regulatory mechanisms ensures the correct activity of EGFR- and FGFR-dependent signaling pathways in the developing eye (Sieglitz, 2013).

Within the RTK signaling pathway, different positive and negative feedback mechanisms have been identified. A prominent negative feedback mechanism is triggered by the secreted protein Argos. Argos expression is induced by RTK activation, and secreted Argos protein can sequester the activating ligand Spitz. In addition, intracellular proteins have been identified to exert a negative feedback function. Sprouty is the most prominent inhibitor of RTK activity and was shown in this study to act downstream of FGFR signaling as well as downstream of EGFR signaling. However, the precise point at which Sprouty intercepts RTK signaling is variable. In the developing fly eye, Sprouty acts upstream of Ras, whereas in the developing wing, Sprouty functions at the level of Raf. An additional negative feedback loop is mediated by the cell surface protein Kekkon, which is specific to EGFR signaling (Sieglitz, 2013).

Positive feedback loops are less frequent and may act through the transcriptional activation of genes that encode activating ligands. This is found in the ventral ectoderm of Drosophila embryos or in follicle epithelium, where the activity of the EGFR pathway is amplified by induction of the expression of its ligand Vein. In addition, the expression of rhomboid may be triggered, which subsequently facilitates the release of activating ligands. Together, these mechanisms ensure a paracrine-mediated amplification of the RTK signal and are thus likely not as effective in regulating RTK activity in single cells (Sieglitz, 2013).

Rau is a previously unidentified positive regulator of RTK signaling that acts within the cell. This study found that Rau function sustains both EGFR and FGFR signaling activity. Rau is a small 51-kD protein that harbors two RA domains, which are found in several RasGTP effectors such as guanine nucleotide–releasing factors. Pull-down experiments demonstrated that Rau preferentially binds GTP-loaded (activated) Ras. The Rau protein is characterized by two RA domains. Although both RA domains are able to bind Ras individually, single RA domains do not show any selectivity toward the GTP-bound form of Ras. Thus, the clustering of two RA domains promotes the selection of RasGTP. In agreement with this notion, it was found that Rau can form dimers or, possibly, multimers. In lysates from embryos, Rau is found in high–molecular weight protein complex, suggesting that it could interact with other components of the RTK signalosome. This way, RA domains are further clustered, and thus, GTP-loaded Ras may be sequestered. In addition, this may also contribute to the clustering of Raf, which is more active in a dimerized state. Moreover, it was recently shown that Ras signaling depends on the formation of nanoclusters at the membrane. This local aggregation may further promote interaction of Ras with Son of sevenless, which can trigger additional activation of the RTK signaling cascade. In addition, Rau harbors a class II PDZ-binding motif, suggesting that Rau can integrate further signals to modulate RTK signaling (Sieglitz, 2013).

The activity of the EGFR and the FGFR signaling cascades is conveyed in part through the transcription factor Pointed. Heterozygous loss of pointed significantly increases the rough eye phenotype evoked by loss of Rau function. Moreover, upon overexpression of the constitutively active PointedP1, rau expression was also increased. In line with this notion, CG8965/rau was also identified in a screen for receptor tyrosine signaling targets. Thus, the data suggest that Rau activation occurs after initial RTK stimulation through direct transcriptional activation through Pointed, which is similar to the activation of the secreted EGFR antagonist Argos (Sieglitz, 2013).

This study has dissected the role of Rau in differentiating glial cells of the fly retina. These glial cells are borne out of the optic stalk and need to migrate onto the eye imaginal disc where some of these cells differentiate into wrapping glial cells upon contacting axonal membranes. The development of these glial cells is under the control of FGFR signaling. Initially, low activity of FGFR signaling in these glial cells is permissive for expression of seven-up, which encodes an orphan nuclear receptor of the COUP-TF (COUP transcription factor) family that suppresses sprouty, but not rau, expression. Activation of Rau requires greater activity of FGFR, which is achieved only through interaction with axons. High activation of FGFR signaling also inhibits seven-up expression and thus relieves the negative regulation of sprouty. This negative regulation of COUP-TFII transcription factors by RTKs is also seen during photoreceptor development in the fly eye and appears to be conserved during evolution (Sieglitz, 2013).

In conclusion, the Rau/Sprouty signaling module provides effective means to sustain a short RTK activation pulse, for example, during cellular differentiation. It is proposed that Rau dimers or multimers assemble a scaffold that favors the recruitment of RasGTP, which then could more efficiently activate the MAPK cascade. Thus, ultimately, Rau may promote the formation of Raf dimers, which might confer robustness and increased signaling intensity. Future studies will reveal the precise conformations and complexes that enable Rau to modulate RTK signaling in fly development (Sieglitz, 2013).

Multifunctional glial support by Semper cells in the Drosophila retina

Glial cells play structural and functional roles central to the formation, activity and integrity of neurons throughout the nervous system. In the retina of vertebrates, the high energetic demand of photoreceptors is sustained in part by Muller glia, an intrinsic, atypical radial glia with features common to many glial subtypes. Accessory and support glial cells also exist in invertebrates, but which cells play this function in the insect retina is largely undefined. Using cell-restricted transcriptome analysis, this study shows that the ommatidial cone cells (aka Semper cells) in the Drosophila compound eye are enriched for glial regulators and effectors, including signature characteristics of the vertebrate visual system. In addition, cone cell-targeted gene knockdowns demonstrate that such glia-associated factors are required to support the structural and functional integrity of neighboring photoreceptors. Specifically, this study shows that distinct support functions (neuronal activity, structural integrity and sustained neurotransmission) can be genetically separated in cone cells by down-regulating transcription factors associated with vertebrate gliogenesis (pros/Prox1, Pax2/5/8, and Oli/Olig1,2, respectively). Further, specific factors critical for glial function in other species are also critical in cone cells to support Drosophila photoreceptor activity. These include ion-transport proteins (Na/K+-ATPase, Eaat1, and Kir4.1-related channels) and metabolic homeostatic factors (dLDH and Glut1). These data define genetically distinct glial signatures in cone/Semper cells that regulate their structural, functional and homeostatic interactions with photoreceptor neurons in the compound eye of Drosophila. In addition to providing a new high-throughput model to study neuron-glia interactions, the fly eye will further help elucidate glial conserved "support networks" between invertebrates and vertebrates (Charlton-Perkins, 2017).

Neuron-glia interactions through the Heartless FGF receptor signaling pathway mediate morphogenesis of Drosophila astrocytes

Astrocytes are critically important for neuronal circuit assembly and function. Mammalian protoplasmic astrocytes develop a dense ramified meshwork of cellular processes to form intimate contacts with neuronal cell bodies, neurites, and synapses. This close neuron-glia morphological relationship is essential for astrocyte function, but it remains unclear how astrocytes establish their intricate morphology, organize spatial domains, and associate with neurons and synapses in vivo. This study characterized a Drosophila glial subtype that shows striking morphological and functional similarities to mammalian astrocytes. The Fibroblast growth factor (FGF) receptor Heartless was demonstrated to autonomously control astrocyte membrane growth, and the FGFs Pyramus and Thisbe direct astrocyte processes to ramify specifically in CNS synaptic regions. It was further shown that the shape and size of individual astrocytes are dynamically sculpted through inhibitory or competitive astrocyte-astrocyte interactions and Heartless FGF signaling. The data identify FGF signaling through Heartless as a key regulator of astrocyte morphological elaboration in vivo (Stork, 2014).

Astrocytes are among the most abundant cell types in the mammalian CNS and fulfill diverse functions in brain development and physiology. In the mature brain, astrocytes buffer ions and pH, metabolically support neurons, and clear neurotransmitters. Astrocytes can sense neuronal activity, react with transient increases of intracellular calcium ion concentration, and in turn modulate neuronal activity. The diverse homeostatic and modulatory roles for astrocytes are essential for neuronal function, and evidence is mounting that this tight physiological relationship between astrocytes and neurons is highly regulated and provides astrocytes with the capacity to exert powerful and dynamic control over neuronal circuits (Stork, 2014).

Astrocytic functions are critically dependent on the intimate spatial relationship between astrocytes and neurons, and accordingly astrocytes exhibit a highly ramified morphology. Primary cellular extensions radiate from the soma of gray matter astrocytes, which then branch into hundreds of increasingly finer cellular processes, ultimately forming a dense meshwork in the brain that associates closely with synapses, neuronal cell bodies, and the brain vasculature. Intriguingly, individual mature mammalian astrocytes occupy unique spatial domains within the brain, apparently 'tiling' through a mechanism akin to dendritic tiling, such that the processes of neighboring astrocytes exhibit very limited overlap. Whether these unique spatial domains are functionally important remains a point of speculation (Stork, 2014).

Despite recent advances in understanding the molecular basis of astrocyte fate specification, control of synapse formation, and neuronal signaling, pathways regulating astrocyte morphogenesis in vivo remain poorly understood. While there appears to be a spatial restriction of astrocyte subtypes to particular regions of the vertebrate CNS, it is not clear whether astrocytes selectively associate with predetermined subsets of neurons. The morphology of individual mammalian astrocytes is quite variable, suggesting that sculpting of their morphology may be stochastic and shaped by cell-cell interactions (Stork, 2014).

This study characterizes a glial cell type in Drosophila remarkably similar to mammalian protoplasmic astrocytes. Drosophila astrocytes dynamically and progressively invade the synaptic neuropil late in embryonic development, associate closely with synapses throughout the CNS, and tile with one another to establish unique spatial domains. The Heartless FGF receptor signaling pathway was identified as a key mediator of astrocyte outgrowth into synaptic regions and the size of individual astrocytes. Through ablation studies, it was demonstrated that individual astrocytes have a remarkable potential for growth, and the establishment of astrocyte spatial domains is mediated by astrocyte-astrocyte inhibitory and/or competitive interactions. This work provides insights into cell-cell interactions governing astrocyte growth in vivo and demonstrates that the requirement for astrocytes is an ancient feature of the nervous system of complex metazoans (Stork, 2014).

Drosophila astrocytes form a highly ramified and dense meshwork of processes that infiltrate the entire neuropil and associate closely with synapses. This close spatial relationship is reminiscent of the mammalian 'tripartite synapse,' thought to be critical for neurotransmitter clearance and the modulation of synaptic activity during complex behaviors. In the L3 VNC, the majority of synapses were in close proximity to astroglial processes, although not directly ensheathed. Nevertheless, using the iGluSnFR reporter, it was demonstrated that local increases in extracellular glutamate readily reached astrocyte membranes, indicating that they are within the functional range of synapses (Stork, 2014).

Functional roles of Drosophila astrocytes also appear well conserved when compared to mammals. The glutamate transporter EAAT1 is expressed in Drosophila astrocytes and is essential for coordinated locomotor activity in larvae and prevention of excitotoxicity in the adult. This study demonstrates astrocyte-specific expression the GABA transporter Gat and partial loss of Gat impeded larval locomotion. GABA transporter inhibitors also impair larval coordinated locomotion, and Manduca and Trichoplusia Gat homologs are high-affinity GABA transporters, supporting the notion that gat-depleted animals experience disruption of GABA neurotransmitter clearance. Despite apparently normal CNS morphology, gat null animals die as late embryos. Astrocytic Gat is therefore essential for viability, and it is proposed that Gat plays a central role for astrocyte-mediated GABA clearance even before animal hatching (Stork, 2014).

Ca2+ microdomain signaling in mammalian astrocytes is emerging as a key mechanism by which astrocytes respond to and regulate neuronal activity. Drosophila cortex glia, cells closely associated with neuronal cell bodies, also exhibit microdomain Ca2+ oscillations, and glial Ca2+ signaling events can modulate fly circadian behavior and seizure activity. Interestingly, this study found Drosophila astrocytes exhibit spontaneous, local Ca2+ transients in vivo and seem to be coupled with respect to Ca2+ signaling: laser-induced injury of a single astrocyte in the larva induced an increase in intracellular calcium in the injured cell, which subsequently spread into neighboring astrocytes (Stork, 2014).

These data taken together argue strongly that Drosophila astrocytes will prove an excellent in vivo system in which to study many fundamental aspects of astrocyte biology and astrocyte-neuron interactions (Stork, 2014).

This study has shown that Drosophila astrocytes are critically important for animal survival. Partial ablation of mouse astrocytes during development also led to death at birth. Interestingly, astrocyte depletion by ~30% in selected spinal cord domains led to atrophy and loss of neuropil and synapses. In Drosophila larvae lacking the majority of astrocytes, gross CNS morphology was surprisingly normal. Therefore, fly astrocytes may not be strictly required for neuronal survival, although earlier ablation or ablations in the adult could yield different results. Alternatively, other subtypes of CNS glia (e.g., ensheathing or cortex glia) might functionally substitute for astrocytes and promote neuronal survival (Stork, 2014).

Depletion of astrocytes from large regions of the mammalian CNS did not lead to a repopulation of these zones by astrocytes from neighboring domains, suggesting that astrocytes possess a high regional specificity and low invasive behavior (Tsai, 2012). However, while dramatic movement of populations of astrocytes was not observed, it is less clear whether astrocytes at the border of astrocyte-depleted regions react more locally with increased growth. Regional astrocyte ablation studies in mammals followed by the use of markers that highlight single-cell astrocyte morphology will be essential to definitively resolve these question (Stork, 2014).

It has been proposed that astrocyte domain organization and association with specific subsets of neurons has an important role in the proper function of neuronal networks. While Drosophila astrocytes are quite stereotyped in cell number and cell body position, the domains of the neuropil covered by astrocyte processes show variability in size and shape. It therefore seems unlikely that individual astrocytes are genetically programmed to associate with particular regions of the brain or specific synapses (Stork, 2014).

Astrocytes appear to harbor a massive growth potential but exert a strong growth-inhibiting effect on one another. First, when adjacent cells are ablated, astrocytes expand their territories while tiling where they are in contact with other astrocytes. Second, when htl or dof mutant clones that failed to infiltrate the neuropil, the space adjacent to these clones was efficiently infiltrated by other astrocytes. Finally, while enhancing Htl signaling increased domain size, neighboring cells still 'tiled' and the overlap of astrocytic domains did not increase noticeably. How tiling of astrocytes occurs remains unclear but could be accomplished through contact-dependent growth inhibition or competition for neuropil growth factors. Nevertheless, based on the multiple lines of evidence presented in this study, it is proposed that astrocyte morphology is shaped dynamically during development by neuron-astrocyte and astrocyte-astrocyte interactions (Stork, 2014).

Finally, while the relative overlap of neighboring astrocytes appears to be higher in Drosophila compared to mammalian astrocytes, it is important to note from a mechanistic perspective that the size of a Drosophila astrocyte is smaller compared to mouse and that the absolute overlap of astrocyte processes in mouse and fly seem comparable. This discovery of tiling behavior in Drosophila suggests that fly and mammalian astrocytes may share common molecular mechanisms by which neighboring cells define their territories (Stork, 2014).

Loss of the FGF receptor Htl, its ligands Pyr and Ths, or the downstream signaling molecule Dof/Stumps blocked the infiltration of astrocyte processes into the neuropil, demonstrating that the Htl signaling pathway is critical for effective astrocytic growth into the synapse-rich neuropil. The level of Htl signaling is also critically involved in the regulation cell and domain size of astrocytes, with increased Htl signaling leading to increased astrocyte volume. Expression data, clonal analysis, and astrocyte-specific rescue experiments all indicate that Htl and Dof function autonomously in glia. Precisely where the FGF ligands Pyr and Ths are generated during development was more difficult to determine. However, based on its expression pattern and the ability to rescue astrocyte outgrowth when expressed in neurons, it is proposed that at least Ths is primarily derived from neurons (Stork, 2014).

Ectopic expression of Pyr or Ths away from the neuropil or astrocytic expression of a constitutively active form of Htl is able to partially restore astrocyte infiltration. These observations suggest a permissive role for the Htl signaling pathway in astroglial growth. However, expression of Pyr or Ths is also able to promote the outgrowth of ectopic astroglial branches outside of the neuropil, indicating that these ligands can provide directional cues for astrocyte outgrowth. Pyr and Ths appear different in their signaling abilities: single neuron expression revealed Pyr was unable to promote extension of astrocyte processes, while Ths drove robust astrocytic process outgrowth, suggesting that the promotion of outgrowth by Ths can act at a short range (Stork, 2014).

How can Pyr and Ths direct astrocyte process growth into the neuropil even when ectopically expressed? FGF signaling is critically dependent on heparan sulfate proteoglycans (HSPGs) in vivo. Two of the four HSPGs in Drosophila, Dally-like and Syndecan, have been reported to be prominently enriched in the embryonic neuropil, where they have been shown to act in Slit-dependent axon guidance. Expression of Sdc in the neuropil was confirmed, ectopic Sdc expression was found to be sufficient to redirect astrocyte membranes outside of the neuropil, and loss of Sdc led to a defect in the ventral migration of astrocyte cell bodies and, to a lesser extent, problems in early neuropil infiltration. Based on these observations it is speculated that Sdc plays a modulatory role in the development of astrocytes by concentrating the FGFs Pyr and Ths in the neuropil to drive directional infiltration even when the ligands are provided ectopically. Finally, Pyr and Ths might act redundantly with additional unidentified neuropil-restricted factors that can provide directional information for astrocytic process outgrowth (Stork, 2014).

ths null mutants showed a slight decrease in the number of astrocytes in late embryos and L3 larvae, while embryonic htlAB42 mutants did not show a reduction in cell counts. sdc mutants also showed a similar slight reduction in total cell number in L3 larvae. These data suggest that astrocytes are generated in the embryo at normal numbers in FGF-pathway mutants but that individual cells might be outcompeted by neighbors during process outgrowth, resulting in death of individual cells. Since it was not possible to uniquely identify the presumptive ventral cell among the dorsally located cells, it is not clear whether the nonmigrating presumptive ventral cells preferentially die or whether cell death is stochastic among all astrocytes. While the mechanism of such adjustment of cell numbers through cellular competition remains poorly understood, it might be based on competition for trophic factors or a more active form of cell killing by 'winning' neighbors. The data deepen understanding of the diverse roles FGF signaling plays in insect glial development, where FGFs have been shown to also regulate glial proliferation, survival, migration and ensheathment of axons, and glial wrapping of FGF2-coated beads in grasshopper (Stork, 2014).

FGF signaling has also been implicated in mammalian astrocyte development. Mammalian FGFs can act as mitogens for glial precursors and potentiate the ability of secreted factors CNTF and LIF to promote astroglial fate in neural progenitors. In addition, FGF application can induce maturation of astroglia in cell culture by controlling morphological stellation in two dimensions and the expression of GFAP and glutamine synthetase. FGF receptors 1-3 are expressed in astrocytes and their precursors. In particular, FGFR3 is highly enriched in the radial precursor cells in the ventricular zone and immature and mature astrocytes and in FGFR3 and other FGF pathway mutants, GFAP expression in astrocytes is perturbed in vivo. Furthermore FGFR1/2 mutants show a reduction in GFAP-positive astrocytes in the cortex and impaired Bergmann glia morphology in the cerebellum. The exact roles of mammalian FGFRs and their ligands in astrocyte ramification, association with neurons and synapses, and establishment of astrocytic domain size, however, remain to be tested. Observations of an essential requirement for FGF signaling in astrocyte development in vivo in Drosophila suggests that a detailed analysis of FGF signaling pathways in mammalian astrocyte development should prove fruitful. FGF signaling is known to be perturbed in glioma, and this study's observations of the key role for FGFs in astrocyte process outgrowth may ultimately provide insight into the highly invasive nature of glioma cells in the brain (Stork, 2014).

Glial glycolysis is essential for neuronal survival in Drosophila

Neuronal information processing requires a large amount of energy, indicating that sugars and other metabolites must be efficiently delivered. However, reliable neuronal function also depends on the maintenance of a constant microenvironment in the brain. Therefore, neurons are efficiently separated from circulation by the blood-brain barrier, and their long axons are insulated by glial processes. At the example of the Drosophila brain, this study addressed how sugar is shuttled across the barrier to nurture neurons. Glial cells of the blood-brain barrier specifically take up sugars and that their metabolism relies on glycolysis, which, surprisingly, is dispensable in neurons. Glial cells secrete alanine and lactate to fuel neuronal mitochondria, and lack of glial glycolysis specifically in the adult brain causes neurodegeneration. This work implies that a global metabolic compartmentalization and coupling of neurons and glial cells is a conserved, fundamental feature of bilaterian nervous systems independent of their size (Volkenhoff, 2015).

Cyclin-dependent kinase 9 is required for the survival of adult Drosophila melanogaster glia

Neuronal and glial progenitor cells exist in the adult Drosophila brain. The primarily glial progenitor cells rely on a microRNA, mir-31a, to inhibit the expression of a predicted E3 ubiquitin ligase, CG16947. Erroneous inheritance of CG16947 by the progeny when the neural progenitor cell divides leads to death of the progeny, however how CG16947 achieves glial cell death is unknown. This study has identified the interacting partner of CG16947 to be Cdk9. Reduction of cdk9 expression in glia causes glial loss; highlighting the importance of cdk9 in mediating the survival of glia. Further, glial loss observed in mir-31a mutants was prevented with adult-specific expression of cdk9 in glia. Biochemical evidence is provided that the binding of CG16947 to Cdk9 causes its degradation. Taken together, this data shows that cdk9 plays a role in the survival of adult glia in the Drosophila brain. Thus, a fine balance exists between mir-31a and CG16947 expression in the progenitor cells that in turn regulates the levels of cdk9 in the progeny. This serves to allow the progenitor cells to regulate the number of glia in the adult brain (Foo, 2017b).

CG16947 is expressed in the progenitor cells and mir-31a serves to limit its expression in the progenitor cells. When this interaction between the microRNA and the CG16947 transcript is abolished, CG16947 is aberrantly translated at higher levels, leading to increased inheritance by the progeny (Foo, 2017b).

This paper shows that depletion of cdk9 alone in adult glia can reduce the number of glia in the adult brain. Thus, cdk9 is necessary for adult glial survival. Additionally, this study shows that in the condition where there are fewer glia in the brain; as in the mir-31a mutants, the adult-specific overexpression of cdk9 in glia prevents glial loss. This shows that it is loss of cdk9 in adult glia that is the likely reason for the glial loss observed in mir-31a mutants (Foo, 2017b).

Using co-immunoprecipitation, evidence is provided that CG16947 binds to and interacts directly with cdk9 leading to its degradation. Taken together, the genetic results and the biochemistry suggest that the subsequent ubiquitination and degradation of cdk9 due to excessive inheritance of CG16947 by the progeny in mir-31a mutants, is the likely cause for glial cell death in these mutants. This study does not exclude the possibility that cdk9 is degraded by other mechanisms, but do show that cdk9 is necessary for glial cell survival. Given the role of cdk9 in controlling RNA-polymerase II-mediated transcription, it is possible that cdk9 works by facilitating the transcription of pro-survival genes (Foo, 2017b).

As an increase in glial number was not observed in the brain when cdk9 was overexpressed in adult glia, it suggests that cdk9 is not sufficient to generate more glial cells unless in aberrant conditions where glia number is reduced, as is in the case of the mir-31a mutants (Foo, 2017b).

Knocking down of CG16947 in progenitor cells did not lead to an increase in glia numbers in the brain. Thus, it suggests that glial numbers are very tightly regulated in the adult brain. The rational being that since high levels of CG16947 inherited by glia from the progenitor cells is detrimental to the survival of glia then, in the wildtype setting, if more glia than necessary are made, then knocking down of CG16947 in progenitor cells, would lead to increased survival of glia and more glia observed. But as more glia is not observed in either setting, when CG16947 is knocked down or cdk9 is overexpressed, it suggests that the number of glia that are made is very tightly regulated and no more than necessary glia are made by the progenitors in the adult central brain of flies (Foo, 2017b).

In mammals, there are several examples of the number of glia in the mature brain being tightly regulated by contact with a target. For instance, mammalian astrocytes are thought to be matched to blood vessels, mediated by the limited secretion of trophic factors from the vasculature. A similar reliance of oligodendrocytes on their target axons for survival has also been observed. This study shows that the number of glia can be in part, regulated from the progenitor cell itself, through controlled expression of CG16947, rather than reliance on the target of the glial cell. These results do not exclude the possibility that glial number in the Drosophila adult brain is regulated by another mechanism (Foo, 2017b).

The mechanism by which mir-31a expression in the progenitor cell is regulated and in turn CG16947 expression is regulated, has yet to be uncovered, however, this study provides evidence that misregulation of this fine balance between mir-31a with CG16947 in the progenitor cells and subsequent excessive degradation of cdk9 in the progeny can alter the number of glia in the adult Drosophila brain. Thus providing evidence for a mechanism by which the progenitor cell itself can regulate the number of progeny that are generated. This study does not exclude the possibility that cdk9 can be regulated in a mir-31a-CG16947-independent manner, but illustrates one way in which cdk9 expression can be regulated in glia (Foo, 2017b).

Chromatin remodeling during the in vivo glial differentiation in early Drosophila embryos

Chromatin remodeling plays a critical role in gene regulation and impacts many biological processes. However, little is known about the relationship between chromatin remodeling dynamics and in vivo cell lineage commitment. This study revealed the patterns of histone modification change and nucleosome positioning dynamics and their epigenetic regulatory roles during the in vivo glial differentiation in early Drosophila embryos. The genome-wide average H3K9ac signals in promoter regions are decreased in the glial cells compared to the neural progenitor cells. However, H3K9ac signals are increased in a group of genes that are up-regulated in glial cells and involved in gliogenesis. There occurs extensive nucleosome remodeling including shift, loss, and gain. Nucleosome depletion regions (NDRs) form in both promoters and enhancers. As a result, the associated genes are up-regulated. Intriguingly, NDRs form in two fashions: nucleosome shift and eviction. Moreover, the mode of NDR formation is independent of the original chromatin state of enhancers in the neural progenitor cells (Ye, 2016).

Antioxidant role for lipid droplets in a stem cell niche of Drosophila

Stem cells reside in specialized microenvironments known as niches. During Drosophila development, glial cells provide a niche that sustains the proliferation of neural stem cells (neuroblasts) during starvation. This study finds that the glial cell niche also preserves neuroblast proliferation under conditions of hypoxia and oxidative stress. Lipid droplets that form in niche glia during oxidative stress limit the levels of reactive oxygen species (ROS) and inhibit the oxidation of polyunsaturated fatty acids (PUFAs). These droplets protect glia and also neuroblasts from peroxidation chain reactions that can damage many types of macromolecules. The underlying antioxidant mechanism involves diverting PUFAs, including diet-derived linoleic acid, away from membranes to the core of lipid droplets, where they are less vulnerable to peroxidation. The study reveals an antioxidant role for lipid droplets that could be relevant in many different biological contexts (Bailey, 2015).

Ensheathing glia function as phagocytes in the adult Drosophila brain

The mammalian brain contains many subtypes of glia that vary in their morphologies, gene expression profiles, and functional roles; however, the functional diversity of glia in the adult Drosophila brain remains poorly defined. This study defines the diversity of glial subtypes that exist in the adult Drosophila brain, show they bear striking similarity to mammalian brain glia, and identify the major phagocytic cell type responsible for engulfing degenerating axons after acute axotomy. Beuropil regions were found to contain two different populations of glia: ensheathing glia and astrocytes. Ensheathing glia enwrap major structures in the adult brain, but are not closely associated with synapses. Interestingly, it was found that these glia uniquely express key components of the glial phagocytic machinery (e.g., the engulfment receptor Draper, and dCed-6), respond morphologically to axon injury, and autonomously require components of the Draper signaling pathway for successful clearance of degenerating axons from the injured brain. Astrocytic glia, in contrast, do not express Draper or dCed-6, fail to respond morphologically to axon injury, and appear to play no role in clearance of degenerating axons from the brain. However, astrocytic glia are closely associated with synaptic regions in neuropil, and express excitatory amino acid transporters, which are presumably required for the clearance of excess neurotransmitters at the synaptic cleft. Together these results argue that ensheathing glia and astrocytes are preprogrammed cell types in the adult Drosophila brain, with ensheathing glia acting as phagocytes after axotomy, and astrocytes potentially modulating synapse formation and signaling (Doherty, 2009).

In an effort to identify distinct morphological subtypes of glial cells in the mature Drosophila brain, the MARCM system was used to generate small clones of glial cells labeled with membrane-tethered GFP. Larvae containing a hs-flipase allele and a wild-type chromosome arm for recombination were subjected to a short heat shock (37°C) and glial cells within clones were visualized by use of the pan-glial driver, repo-Gal4. The analysis of glial subtype morphology was focused mainly on the adult antennal lobe region owing to its well defined histology and accessibility to genetic manipulation. In the adult brain clones were identified resembling each of the three main types of glial cells found in embryos and larvae: (1) cortex glia, which resided outside the neuropil in regions housing neuronal cell bodies, ramified dramatically to surround individual cell bodies; (2) surface glia, which appeared as large flat cells enveloping the surface of the brain, did not extend any processes into the brain; and (3) neuropil glia, which were closely associated with the neuropil, extended membranes into synaptic regions, and surrounded large bundles of axons. As in the embryo and larva, glial cell bodies were not found within the neuropil, rather they resided at the edge of the neuropil (neuropil glia), in the cortex (cortex glia), or at the surface of the brain (surface glia) (Doherty, 2009).

Interestingly, the single-cell resolution provided by MARCM analysis allowed further subdivision of neuropil glia into two distinct morphological classes, 'ensheathing glia' and 'astrocytic glia.' Ensheathing glia appeared as flattened cells that lined the borders of the neuropil and subdivided regions of the brain by isolating neuropil from the surrounding cortex. Within the antennal lobe, ensheathing glial membranes surrounded individual glomeruli (the functional units of the antennal lobe) but did not extend into the synaptic regions of the glomeruli. In addition, an astrocyte-like cell type was identified that extended membrane processes deeply into the neuropil and ramified profusely in synaptic-rich regions. This latter cell type is referred to as the fly 'astrocyte', based on its striking morphological similarity to mammalian astrocytes, as well as the conserved expression of a number of molecular markers used to identify astrocytes in the mammalian brain. Mammalian astrocytes remove excess amounts of extracellular glutamate through the high-affinity excitatory amino acid transporters (EAATs), GLAST and GLT-1, which transports the glutamate into glial cells where it is then converted into glutamine by glutamine synthetase. It was found that Drosophila astrocytes also express the transporter EAAT1. While the adult antennal lobe was used as the primary model tissue in this study, the morphological glial subtypes described above were observed in all brain regions examined, suggesting that the results are generally applicable to glial populations throughout the adult Drosophila brain (Doherty, 2009).

Attempts were made to identify Gal4 driver lines that would allow unique labelling and and manipulation of these glial populations, with a major focus being to genetically subdivide neuropil glia (i.e., ensheathing glia versus astrocytes). To accomplish this, UAS-mCD8::GFP was crossed to a previously described collection of embryonic and larval glial drivers (Ito, 1995), as well as a number of drivers generated for this study. The adult antennal lobe was studied to examine the morphology and spatial distribution of cell types marked by these drivers in a background with glial nuclei ({alpha}-Repo) and the neuropil ({alpha}-nc82) also labeled. The repo-Gal4 driver labeled all Repo+ glial subtypes in the adult brain, as evidence by {alpha}-Repo immunostaining in the nuclei of GFP+ cells. Membrane processes from Repo+ cells are found throughout the adult brain, and together they constitute the diverse collection of glial subtypes identified in single-cell MARCM analysis. Upon examination of a single glomerulus within the antennal lobe, membranes from Repo+ cells were found to both surround and invade glomeruli. All GFP expression in the adult brain driven by the repo-Gal4 driver can be suppressed by coexpression of Gal80 (a Gal4 inhibitor) under control of the repo promoter (repo-Gal80), arguing that repo-Gal80 can efficiently block Gal4-mediated activation of UAS-reporters in all adult brain glia (Doherty, 2009).

Two drivers, mz0709-Gal4 and alrm-Gal4, appeared to show very specific expression in ensheathing glia and astrocytes, respectively. Glial processes labeled by mz0709-Gal4 were found at the edge of the antennal lobe and extended deeply into the neuropil region. These flattened glial processes surrounded, but did not invade, individual glomeruli, and did not extend into the cortex region. With the exception of variable expression in a small number of neurons, all mz0709-Gal4-induced expression was suppressed by repo-Gal80, indicating that mz0709-Gal4 is largely specific to ensheathing glia. The generation of MARCM clones labeled with the mz0709-Gal4 driver resulted in the consistent labeling of ensheathing glia, but not astrocytes, within the antennal lobes. Reciprocally, alrm-Gal4 was found to be expressed exclusively in astrocytes. All cellular processes from cells labeled with alrm-Gal4 extended into the neuropil, showed a highly branched or tufted morphology, invaded individual glomeruli, and all alrm-Gal4-driven expression was suppressed by repo-Gal80. Additionally, single cell MARCM clones were found labeled with the alrm-Gal4 driver resulted in the consistent labeling of astrocytes, but not ensheathing glia. Together, these drivers are excellent tools to manipulate and functionally distinguish different subtypes of glia in the adult Drosophila brain (Doherty, 2009).

What are the functional roles for each glial subtype in the adult brain? Is each subtype responsible for a unique collection of tasks, or are all glial subtypes functionally equivalent? As a first step to determining the in vivo functional differences between adult brain glial subtypes the cell autonomy of glial phagocytic function was explored. Severing olfactory receptor neuron (ORN) axons by surgical ablation of maxillary palps leads to axon degeneration (termed Wallerian degeneration), recruitment of glial membranes to fragmenting axons, and glial engulfment of axonal debris. These glial responses are mediated by Draper, the Drosophila ortholog of the C. elegans cell corpse engulfment receptor cell death defective-1 (CED-1). In draper null mutants, glia fail to extend membranes to degenerating ORN axons and axonal debris is not removed from the CNS. Thus, Draper function should be autonomously required in phagocytic glial subtypes and Draper expression is predicted to act as a molecular marker for glial cells capable of performing engulfment functions (Doherty, 2009).

To define the precise cell types that express Draper, all glial membranes were labeled with mCD8::GFP driven by repo-Gal4, stained with α-Draper antibodies, and assayed for colocalization of Draper and GFP. Extensive overlap of Draper and GFP was found in this background. Draper and GFP signals overlapped at the edge of the neuropil, in membranes surrounding antennal lobe glomeruli, and in all cortex glia. This labeling was specific to Draper since expression of a UAS-draperRNAi construct with repo-Gal4 led to the elimination of all Draper immunoreactivity in the adult brain. Thus, the entire population of cortex glia appear to express Draper and are likely to be phagocytic. However, cortex glia do not extend membranes into the antennal lobe neuropil, even after ORN axon injury. Therefore, cortex glia are not likely responsible for clearing severed ORN axonal debris from the antennal lobe neuropil (Doherty, 2009).

Interestingly, when mCD8::GFP was driven by mz0709-Gal4 extensive overlap of Draper and GFP was observed in neuropil-associated ensheathing glia. A high-magnification view of the antennal lobe revealed Draper and mz0709-Gal4 labeled membranes colocalizing and surrounding, but not innervating individual glomeruli. Moreover, expression of UAS-draperRNAi in ensheathing glia with mz0709-Gal4 led to a dramatic reduction in Draper immunoreactivity in the neuropil, but the weaker Draper immunoreactivity in the cortex remained unchanged. Conversely, no overlap was observed between Draper and GFP when astrocytic membranes were labeled using the alrm-Gal4 driver. Furthermore, driving the expression of UAS-draperRNAi in astrocytes had no obvious effect on Draper expression in the brain. These results indicate that Draper is expressed in cortex glia and ensheathing glia but not in astrocytes (Doherty, 2009).

The specific expression of Draper in antennal lobe ensheathing glia suggests that this glial subset is the phagocytic cell type responsible for engulfing degenerating axonal debris after ORN axotomy. To explore this possibility, it was asked whether ensheathing glia or astrocytes extend membranes to severed axons after injury, and in which cell type Draper was required for clearance of axonal debris from the CNS. To assay extension of glial membranes to severed axons, glial membranes were labeled with mCD8::GFP, maxillary palp axons were severed, and colocalization of Draper and GFP was assayed in glomeruli housing severed ORN axons. Within 1 d after injury, Repo+ glial membranes were found to localize to glomeruli housing severed maxillary palp axons and these membranes were decorated with Draper immunoreactivity. Similarly, mz0709+ glial membranes also localized to severed axons and colocalized with intense Draper immunoreactivity 1 d after injury. Knockdown of Draper with UAS-draperRNAi using repo-Gal4 or mz0709-Gal4 completely suppressed the recruitment of both Draper and glial membranes to severed axons. In contrast, when astrocyte membranes were labeled with GFP no colocalization of GFP and Draper immunoreactivity was observed 1 d after axotomy. In addition, knockdown of Draper in astrocytes with UAS-draperRNAi did not suppress the recruitment of Draper to severed axons. In an effort to identify any indirect role for astrocytes during the injury response, the morphology of astrocytes was examined both before and after injury to determine whether they exhibited any overt changes in morphology or retracted their membranes from the site of injury to accommodate the recruitment of ensheathing glial membranes. However, no obvious changes were detected in morphology or in the positions of the astrocyte glial cells in response to axon injury. Together, these data indicate that Draper is required in ensheathing glia for recruitment of glial membranes and accumulation of Draper on severed ORN axons, and suggest that Drosophila astrocytic glia do not undergo any dramatic changes in morphology in response to ORN axotomy (Doherty, 2009).

From the above data it is predicted that ensheathing glia would act as phagocytes to engulf degenerating ORN axonal debris from the CNS. To test this a subset of maxillary palp ORN axons was labeled with mCD8::GFP using the OR85e-mCD8::GFP transgene, Draper function was knocked down in glial subsets using subset-specific driver lines, maxillary palp ORN axons were severed, and clearance of axons was assayed 5 d after injury. GFP-labeled axons were first severed in control animals with each driver and GFP+ axonal debris was found to be efficiently cleared from the CNS within 5 d after injury, confirming that glial phagocytic function is not affected in the driver lines. Strikingly, RNA interference (RNAi) knockdown of Draper using UAS-draperRNAi in a background with repo-Gal4 or mz0709-Gal4 completely blocked clearance of GFP-labeled axonal debris from the CNS, while RNAi knockdown of Draper in astrocytes with alrm-Gal4 had no effect on axon clearance. Thus, Draper is required autonomously in ensheathing glia for the clearance of degenerating ORN axonal debris from the CNS. In addition, knockdown of Draper in ensheathing glia with mz0709-Gal4 had no measurable effect on Draper expression in cortex glia, arguing that cortex glia are not capable of compensating for the loss of phagocytic activity in ensheathing glia during the clearance of axonal debris from the antennal lobe neuropil after axotomy. From these data on morphogenic responses to injury and phagocytic function, it is concluded that astrocytic, cortex, and ensheathing glia represent functionally distinct subsets of glial cells in the adult Drosophila brain (Doherty, 2009).

The Draper signaling pathway is a central mediator driving glial engulfment of neuronal cell corpses, and axons undergoing Wallerian degeneration in Drosophila. These studies show that within the adult brain neuropil, ensheathing glial cells are the only cell type that expresses Draper. Draper expression overlaps precisely with ensheathing glial membranes (when labeled with GFP driven by the ensheathing glia-specific driver mz0709-Gal4), and RNAi for draper in ensheathing glia leads to the elimination of Draper immunoreactivity in the neuropil. Similarly, it was found that dCed-6, the fly ortholog of C. elegans CED-6, a PTB-domain binding protein that functions genetically downstream of worm CED-1, is expressed in the adult brain in a pattern indistinguishable from Draper. dCed-6 immunoreactivity is strongly reduced in the neuropil through mz0709-Gal4-mediated knockdown (with UAS-dCed-6RNAi), and eliminated from the entire brain when dCed-6RNAi treatment is performed with the pan-glial driver repo-Gal4. Thus, in the adult brain dCed-6 appears to be expressed exclusively in glia, including cortex glia and ensheathing glia of the neuropil (Doherty, 2009).

Components of the Draper signaling pathway are required specifically in ensheathing glia for glial membrane recruitment to severed ORN axons, and clearance of degenerating axonal debris from the brain. Knocking down either draper or shark in ensheathing glia is sufficient to block recruitment of glial membranes and Draper to severed axons and clearance of degenerating axonal debris from the CNS. It is suspected that dCed-6 is also required in ensheathing glia for a number of reasons. First, dCed-6 and Draper show perfect overlap in expression in the neuropil, and Draper is only expressed in ensheathing glia. Second, ensheathing glia are recruited to degenerating axon injury, and dCed-6, like Draper, is specifically recruited to degenerating maxillary palp ORN axons after maxillary palp ablation. Third, dCed-6 expression is dramatically increased in ensheathing glia surrounding the antennal lobe after antennal ablation, similar to what has been found for Draper. Surprisingly, RNAi knock down of dced-6 in ensheathing glia (with mz0709-Gal4) failed to suppress the recruitment of Draper to severed ORN axons or the clearance of axonal debris from the brain. It is suspected that this is because mz0709-Gal4-mediated knockdown of dced-6 is incomplete in ensheathing glia, based on reduced but not eliminated staining in this background. Nevertheless, the observations that dCed-6 is localized with Draper in immunostains, is required in glia by RNAi knockdown with repo-Gal4, and that a null allele of dced-6 genetically interacts with draper null mutations in axon engulfment, argues strongly for a role for dCed-6 in ensheathing glia (Doherty, 2009).

Moreover, knockdown of draper in ensheathing glia, or suppressing glial engulfing activity by blocking endocytic function with Shibirets fully suppresses the recruitment of Draper to severed axons and clearance of degenerating axonal debris from the brain. In contrast, astrocytes do not express Draper (or dCed-6), fail to respond morphologically to axon injury, and knockdown of draper in astrocytes has no effect on the recruitment of Draper to severed axons or clearance of axonal debris from the CNS. Such a separation of phagocytic function in neuropil glial cells suggests similarities to the assigned functional roles in mammalian glia, with ensheathing glia as resident phagocytes, engulfing the majority or all axonal debris and astrocytes perhaps playing a less important role in phagocytosis of degenerating axons (Doherty, 2009).

It is concluded that ensheathing glia are the phagocytes of the central brain, responsible for engulfing degenerating ORN axons after axotomy. Based on their expression of Draper and dCed-6 it is proposed that cortex glia play a similar role in the cortex, perhaps engulfing degenerating axons in this tissue, or cell corpses generated during neuronal development or after brain injury. Drosophila astrocytes, in contrast, are in close association with synapse rich regions of the brain and it is speculated they likely play an important role in neural circuit and synapse physiology. This work represents the first functional dissection of glial subtypes in the central brain of adult Drosophila, and lays the foundation for future functional studies of these diverse classes of glia (Doherty, 2009).

Loss of focal adhesions in glia disrupts both glial and photoreceptor axon migration in the Drosophila visual system

Many aspects of glial development are regulated by extracellular signals, including those from the extracellular matrix (ECM). Signals from the ECM are received by cell surface receptors, including the integrin family. Previous studies have shown that Drosophila integrins form adhesion complexes with Integrin-linked kinase and talin in the peripheral nerve glia and have conserved roles in glial sheath formation. However, integrin function in other aspects of glial development is unclear. The Drosophila eye imaginal disc (ED) and optic stalk (OS) complex is an excellent model with which to study glial migration, differentiation and glia-neuron interactions. The roles of the integrin complexes was studied in these glial developmental processes during OS/eye development. The common β subunit βPS and two α subunits, αPS2 and αPS3, are located in puncta at both glia-glia and glia-ECM interfaces. Depletion of βPS integrin and talin by RNAi impaired the migration and distribution of glia within the OS resulting in morphological defects. Reduction of integrin or talin in the glia also disrupted photoreceptor axon outgrowth leading to axon stalling in the OS and ED. The neuronal defects were correlated with a disruption of the carpet glia tube paired with invasion of glia into the core of the OS and the formation of a glial cap. These results suggest that integrin-mediated extracellular signals are important for multiple aspects of glial development and non-autonomously affect axonal migration during Drosophila eye development (Xie, 2014).

This study found that OS glia express integrin complexes that play a role in the development of the glia and axons of the ED and OS. βPS integrin is located in puncta at the glial membrane and associates with Talin and ILK. These focal adhesion markers are found between the perineural glia (PG) and ECM, plus at the interfaces of the PG-CG (carpet glia) and CG-CG layers. A different distribution was found for the αPS2 and αPS3 integrins, which were concentrated at the periphery and interior of the OS, respectively. The results from RNAi-mediated knockdown revealed that these complexes play important roles in OS glial development, as knockdown led to disruption of PG and CG morphology. Specifically, the loss of βPS integrin or talin caused PG to aggregate in the distal half of the OS, resulting in an accumulation of glia in the OS. The PG formed clusters instead of a surrounding monolayer, suggesting that PG make integrin-mediated associations that maintain their distribution (Xie, 2014).

The PG migrate between the CG and the basal ECM, and loss of focal adhesions led to a disruption in PG migration, suggesting that integrin complexes on one or both surfaces play a role in mediating glia migration. The αPS2/βPS heterodimer binds ligands containing the tripeptide RGD sequence and αPS3/βPS binds laminins, so either or both could mediate adhesion of the PG to the ECM. However, it appears that depletion of the integrin complex in apposing glial layers is necessary to disrupt glial migration into the OS, as MARCM clones within the PG alone did not disrupt migration. Integrin function is conserved in mediating glial cell migration either on ECM or neighboring glial surfaces. For example, vertebrate glial studies found that integrins are involved in astrocyte, oligodendrocyte precursor and Schwann cell migration on various ECM molecules. Loss of β1-integrin in Bergmann glia leads to mislocalization, ectopic migration and disruption of process growth within the vertebrate cerebellum. ILK and CDC42 within Bergmann glia are required for the β1-integrin-dependent control of process outgrowth (Xie, 2014).

Reduction of focal adhesions disrupted the CG sheath and the integrity of the blood-nerve barrier, suggesting that maintenance of the CG tube also requires integrin-mediated adhesion. The disruption of the CG tube is similar to observations made in vertebrates, in which glial tubes are necessary for chain migration of neuroblasts along the rostral migratory stream; β1-integrin plays a role in both chain migration and maintenance of the glial tubes. However, the link between the integrin complex and the formation or stabilization of the CG tube is currently unknown (Xie, 2014).

The integrin complex appears to play a limited role in the migration of the WG into the OS. After knockdown of βPS or talin, WG with normal bipolar membrane processes were observed and WG mys1 MARCM clones had morphologies similar to control clones, suggesting that integrin signaling is not required for migration in differentiated WG. TEM analysis suggests that the WG failed to properly ensheath and segregate the bundles of photoreceptor axons, a phenotype consistent with that observed in vertebrate glia, although it is also possible that the lack of WG ensheathment is a secondary effect of axon stalling (Xie, 2014).

Loss of integrin complexes resulted in a failure of photoreceptor axons to exit the ED, navigate the OS or correctly target the optic lobe. Previously, blocking glial migration from the OS using dominant-negative Ras1 (Ras85D -- FlyBase) resulted in photoreceptor axons stalling in the ED but not the OS. The phenotype suggested that photoreceptor axons require physical contact with retinal glia to exit the ED. However, this mechanism does not seem to apply to the stalling phenotype observed with knockdown of the integrin complex. In the majority of samples, glia were still present in the ED and around the axonal stalling region, suggesting that the axon stalling phenotypes are likely to be due to a different mechanism (Xie, 2014).

It is possible that axon stalling results from a combination of glial changes affecting the multiple subtypes of OS glia and disruption of both the βPS/αPS2 and βPS/αPS3 adhesion complexes. Only the simultaneous loss of both αPS2 and αPS3 triggered the axonal phenotypes. Similarly, knockdown of βPS or talin within individual glial subtypes does not trigger axon stalling, whereas disruption of adhesion complexes in all glial subtypes does. It might be that only the repo-GAL4 driver is sufficiently strong or expressed early enough for the effective knockdown of integrin or talin to trigger the axon stalling phenotypes. However, the axon stalling phenotype can be effectively produced by delaying the expression of the RNAi with the repo-GAL4 driver until the second instar, suggesting that early expression is not key. Overall, the results suggest that it is the combined loss of the focal adhesion complex in multiple glial layers that led to the axon stalling phenotype, although the underlying mechanism is not known. It is possible that the simultaneous aggregation of the stalled PG and disruption of the CG sheath triggers axon stalling by allowing ectopic PG to enter the center of the OS or form the glial cap. The ectopic PG within the axon stalling area and in the glial cap were likely to be PG given their expression of LanB2, Apt and the lack of the WG Gli-lacZ marker. Normally, the PG migrate into the ED and differentiate into WG in the presence of photoreceptor axons. However, in the RNAi-treated OS many of the Apt-positive glia also expressed Gli-lacZ, suggesting a change in the normal differentiation pathway, perhaps owing to the premature and ectopic contact of the PG with the photoreceptors within the OS. Loss of integrins throughout the entire glial population could also lead to global changes to the ECM, as loss of integrins can alter the deposition of ECM components during epithelial morphogenesis. Although loss of integrins in the PNS does not lead to changes in the neural lamella of the peripheral nerve, it is possible that ECM changes in terms of structural integrity or the ability to recruit protein components could result in the multiple glial morphological changes (Xie, 2014).

In summary, this study has shown that glia in the OS and ED express integrins and Talin, through which they receive external signals important for PG migration, organization and CG barrier formation. The combined impact of integrin complexes on the morphology and development of both glial layers is crucial for proper axonal outgrowth through the OS and targeting in the brain (Xie, 2014).

Cell death triggers olfactory circuit plasticity via glial signaling in Drosophila

The Drosophila antennal lobe is organized into glomerular compartments, where olfactory receptor neurons synapse onto projection neurons. Projection neuron dendrites also receive input from local neurons, which interconnect glomeruli. This study investigated how activity in this circuit changes over time when sensory afferents are chronically removed in vivo. In the normal circuit, excitatory connections between glomeruli are weak. However, after receptor neuron axons projecting to a subset of glomeruli were chronically severed by removal of antennae, it was found that odor-evoked lateral excitatory input to deafferented projection neurons was potentiated severalfold. This was caused, at least in part, by strengthened electrical coupling from excitatory local neurons onto projection neurons, as well as increased activity in excitatory local neurons. Merely silencing receptor neurons was not sufficient to elicit these changes, implying that severing receptor neuron axons is the relevant signal. When the neuroprotective gene Wallerian degeneration slow (WldS; Hoopfer, 2006) was expressed in receptor neurons before severing their axons, this blocked the induction of plasticity. Because expressing WldS prevents severed axons from recruiting glia, this result suggests a role for glia. Consistent with this, it was found that blocking endocytosis in ensheathing glia blocked the induction of plasticity. In sum, these results reveal a novel injury response whereby severed sensory axons recruit glia, which in turn signal to central neurons to upregulate their activity. By strengthening excitatory interactions between neurons in a deafferented brain region, this mechanism might help boost activity to compensate for lost sensory input (Kazama, 2011).

The results demonstrate that when all the ORN afferents to a subset of glomeruli are removed, excitatory interactions between glomeruli become stronger. As a result, deafferented PNs acquire robust responses to odors. Whereas normal PNs respond selectively to different odor stimuli, deafferented PNs respond nonselectively. This presumably reflects the fact that each excitatory LN (eLN; excitatory input to projection neurons) arborizes in most or all glomeruli. Thus, these PNs likely pool indirect excitatory input from all surviving ORNs (Kazama, 2011).

The key finding of this study is that that removing ORN input causes an upregulation of excitatory connections between glomeruli. Previously, it was shown that overstimulating one ORN type causes an upregulation of inhibitory input to a glomerulus (Sachse, 2007). Both of these phenomena may be seen as forms of compensatory plasticity. Compensatory plasticity also occurs in the mammalian olfactory bulb at several synaptic sites (Kazama, 2011).

Silencing electrical activity in ORNs was not sufficient to induce the same functional changes produced by severing ORN axons. This implies that the trigger is not the loss of electrical activity, but rather a molecular signal that is produced by severed axons. Mis-expressing WldS in ORNs blocks induction, and this implies that WldS suppresses the signal that severed axons produce. Suppressing endocytosis in ensheathing glia also blocks induction. This suggests that the signal produced by severed axons acts on glial receptors that require endocytosis for signal transduction. It is interesting that blocking endocytosis in astrocytes had no effect, because astrocytes interact with neurons in other systems. It is possible that astrocytes are involved in this process, but astrocytic endocytosis is not required (Kazama, 2011).

It is notable that both the manipulations that blocked the induction of plasticity (mis-expressing WldS in ORNs, or blocking endocytosis in ensheathing glia) also block the recruitment of ensheathing glia into deafferented glomeruli after ORNs are removed. This would appear to suggest that the same signal triggers both neural plasticity and morphological changes in glia. However, these signaling cascades clearly diverge: the recruitment of glial membranes to degenerating neurons is blocked by mutating the glial transmembrane receptor draper, whereas draper is not required for the plasticity described in this study. Interestingly, removing only one antenna was not sufficient to induce plasticity in glomerulus VM2 PNs. This manipulation kills half the ORNs that target these PNs. It should be noted that removing both palps kills fourfold fewer ORNs than removing one antenna, and this manipulation also affects fewer glomeruli, yet this was sufficient to induce plasticity in palp PNs. Removing both palps is also sufficient for glial mobilization and phagocytosis in the palp glomeruli (Doherty, 2009). The current results argue that the relevant factor is not the total number of afferents that are killed, but the proportion of live and dead axons in a given glomerulus. However, it also seems that killing all the ORNs that target a single glomerulus is not sufficient. This conclusion arises from the finding that removing the ipsilateral antenna did not produce potentiation in glomerulus V PNs, which receive strictly ipsilateral antennal input. This result implies that some minimum number of glomeruli must be completely deafferented to trigger the described phenomenon (Kazama, 2011).

The results indicate that after some ORNs are chronically removed, several changes occur in the antennal lobe circuit over time. First, depolarization propagates more effectively from eLNs to PNs. This could reflect increased gap junctional conductance from eLNs onto PNs. However, the possibility cannot be excluded that it is the result of a change in the intrinsic properties of eLNs that produces better propagation of voltages from the eLN soma to the site of the eLN-PN gap junctions. In this latter scenario, there would not necessarily be a change in gap junction conductance. Because good voltage clamp in eLNs cannot be achieved, these alternatives could not be evaluated directly, but two pieces of evidence argue for a change in the gap junction itself. First, the gap junction subunit composition of these electrical connections is evidently changed, because it was observed that electrical coupling from eLNs onto PNs is no longer completely dependent on the ShakB.neural subunit. Whereas in normal flies odor-evoked lateral excitation is abolished by the shakB2 mutation, which eliminates ShakB.neural, odor-evoked lateral excitation is not abolished in mutant antennal PNs after chronic antennal removal. Second, no significant change was found in any intrinsic properties of eLNs, including input resistance, resting potential, or excitability (Kazama, 2011).

A second change that occurs in chronically deafferented PNs is that spontaneous membrane potential fluctuations are larger in these PNs compared with acutely deafferented PNs. This may result from the increased input from eLNs onto PNs (Kazama, 2011).

A third change is that odors elicit stronger depolarization in eLNs. The intrinsic excitability of eLNs does not significantly increase, and therefore this change is likely caused by increased synaptic drive to eLNs. This potentiated synaptic drive may originate from PNs: because odor responses in deafferented PNs become larger after the induction of plasticity, and because PNs make chemical as well as electrical synapses onto eLNs, a net increase in the synaptic drive that PNs provide onto eLNs would be expected. In addition, it is possible that ORN-to-eLN synapses are potentiated (Kazama, 2011).

In sum, the net effect of these changes is to produce more robust activity in chronically deafferented PNs, compared with acutely deafferented PNs. These findings also help explain why plasticity is expressed globally rather than locally: if eLNs are responding more robustly to odors, and each eLN innervates all glomeruli, then this increased excitation should propagate across the antennal lobe (Kazama, 2011).

Whereas normal PNs are selective for odor stimuli, the potentiated odor responses of deafferented PNs are comparatively nonspecific. This presumably reflects the fact that each eLN arborizes in most or all glomeruli and so likely pools input from all surviving ORN types. Nevertheless, the odor responses of deafferented PNs may still be useful from the perspective of higher olfactory brain regions. Because acutely deafferented PNs regain normal levels of activity over time, this type of plasticity should tend to restore normal levels of activity in higher olfactory regions. This might help maintain the sensitivity of these regions to sensory signals, or maintain tropic support to these regions (Kazama, 2011).

More broadly, it is speculated that the phenomenon describe in thes study might reflect a general injury response in the Drosophila nervous system, and perhaps also a phenomenon that occurs during normal nervous system development. By triggering the upregulation of specific interactions between surviving neurons following the death of other neurons, this mechanism might help increase the number of neurons that are driven by active afferents. This could be a generally useful adaptation to neuronal death because it should tend to maintain total neural activity within a normal dynamic range (Kazama, 2011).

The reorganization of central sensory representations following changes in sensory input is generally thought to reflect changes in the strength of chemical synapses. The results suggest that central electrical synapses can also be persistently altered following sensory deafferentation. It is well known that neuromodulators can produce short-term changes in the strength of electrical synapses, as illustrated by studies in the vertebrate retina and crustacean stomatogastric ganglion. There are fewer examples of long-term changes in electrical synapse strength, but a growing literature suggests that this may be a fundamental mechanism of neural plasticity (Kazama, 2011).

The reorganization of central sensory representations following sensory deafferentation is sometimes assumed to be triggered by reduced electrical activity, not cell death. However, there is growing evidence that changes in electrical activity may produce synaptic plasticity via signaling pathways that are also linked to injury and inflammation. Thus, changes in electrical activity can produce synaptic plasticity by 'co-opting' signaling systems that are involved in injury responses. The results show that, in the Drosophila antennal lobe, some functional rearrangements following deafferentation can be specific responses to cell death signals, and are not necessarily induced by electrical silencing. In this study, reduced electrical activity was disambiguated from cell death because genetic tools were used to create 'undead' severed axons. The results are reminiscent of studies in vertebrates showing that sensory afferent death can produce changes in target brain regions that are not mimicked by electrical silencing using pharmacological manipulations (Kazama, 2011).

Finally, these findings provide a new window on neural-glial interactions. In mammals, there is good evidence that glia can modulate synaptic transmission and neural excitability. In both mammals and in Drosophila, glia also play important roles following injury. In particular, there are many instances of sensory afferent injury causing morphological changes in glia and glial proliferation in target brain regions . However, it is not entirely clear how such glial responses might affect neuronal physiology and sensory codes in these brain regions. The results illustrate specific cellular and synaptic changes in a sensory circuit that result from glial responses to sensory afferent injury. More broadly, the results illustrate the power of Drosophila as a genetically tractable model for studying neural-glial interactions in vivo (Kazama, 2011).

Engulfing action of glial cells is required for programmed axon pruning during Drosophila metamorphosis

Axon pruning is involved in establishment and maintenance of functional neural circuits. During metamorphosis of Drosophila, selective pruning of larval axons is developmentally regulated by ecdysone and caused by local axon degeneration. Previous studies have revealed intrinsic molecular and cellular mechanisms that trigger this pruning process, but how pruning is accomplished remains essentially unknown. Detailed analysis of morphological changes in the axon branches of Drosophila mushroom body (MB) neurons revealed that during early pupal stages, clusters of neighboring varicosities, each of which belongs to different axons, disappear simultaneously shortly before the onset of local axon degeneration. At this stage, bundles of axon branches are infiltrated by the processes of surrounding glia. These processes engulf clusters of varicosities and accumulate intracellular degradative compartments. Selective inhibition of cellular functions, including endocytosis, in glial cells via the temperature-sensitive allele of shibire both suppresses glial infiltration and varicosity elimination and induces a severe delay in axon pruning. Selective inhibition of ecdysone receptors in the MB neurons severely suppresses not only axon pruning but also the infiltration and engulfing action of the surrounding glia. These findings strongly suggest that glial cells are extrinsically activated by ecdysone-stimulated MB neurons. These glial cells infiltrate the mass of axon branches to eliminate varicosities and break down axon branches actively rather than just scavenging already-degraded debris. It is therefore proposed that neuron-glia interaction is essential for the precisely coordinated axon-pruning process during Drosophila metamorphosis (Awasaki, 2004).

The dorsal and medial axon branches of the larval γ neurons have many varicosities along its trajectory that are clearly visualized when a single γ neuron is labeled with cytoplasmic-GFP (cGFP) reporter combined with the mosaic analysis with a repressible cell marker (MARCM) system and the enhancer-trap strain GAL4-201Y. These axon branches are also labeled strongly with the anti-Synaptotagmin antibody, which detects synaptic vesicle protein accumulated in the presynaptic sites. Thus, the larval γ neurons seem to be extensively interconnected with other neurons through synapses. Because synaptic terminals are condensed in the varicosities, the fate of these varicosity structures were first examined during the time course of axon pruning (Awasaki, 2004).

The number of varicosities on a dorsal axon branch was counted at various developmental stages. A single branch contained 20.7 and 21.4 varicosities on average in the brains of late third-instar larvae (L3) and in pupae just after puparium formation (0 hr APF), respectively. The number of varicosities was reduced by approximately 35% at 6 hr APF (an average of 13.6 varicosities per axon branch). To compare the timing of local axon degeneration and reduction of varicosity numbers, the frequency of disconnected axons was simultaneously examined. Only a very few axon branches were disconnected at 6 hr APF (2 of 60 observed axons) (Awasaki, 2004).

At 12 hr APF, approximately 90% of varicosities disappeared (an average of 2.0 varicosities per branch) and 75% of axons were disconnected (30 of 40). At 18 hr APF, more than 95% of varicosities disappeared (an average of 0.9 varicosities per branch) and 90% of axons were disconnected (48 of 53). In many cases, distal portions of disconnected axons remained at 12 hr APF, and only distal tips of axons frequently remained at 18 hr APF, suggesting that the shaft region of the axon branch tends to degenerate first. At 12 hr and 18 hr APF, the number of varicosities was drastically reduced even on the axons that were not yet disconnected (an average 2.8 [n = 10] and 1.4 [n = 5] varicosities per branch, respectively). These data suggest that disappearance of varicosities precedes local axon degeneration (Awasaki, 2004).

Pruning of axons is triggered by ecdysone through the complex of ecdysone receptor type B1 (EcR-B1) and Ultraspiracle (USP). If the disappearance of varicosities is associated with programmed axon pruning, the disappearance should be suppressed by inhibition of ecdysone signaling. To address this, axon branches of the clones of usp mutant γ neurons were examined. Both disappearance of varicosities and axon disconnection were significantly suppressed. Thus, these phenomena were regulated cell autonomously by the ecdysone-induced stimulation of γ neurons (Awasaki, 2004).

There are more than 2000 neurons that form the larval MB, and these results have indicated that disappearance of the varicosities proceeds gradually during the first 18 hr of the pupal stage. How do the varicosities disappear in these masses of axon branches? Do they disappear randomly, one by one, or in a coordinated process? To examine this, the detailed structure of the dorsal MB lobe was observed by using the enhancer-trap strain GAL4-201Y and the anti-Fasciclin II (FasII) antibody, both of which label axons of γ neurons from the larva to adult stage (Awasaki, 2004).

In the dorsal lobe of late larvae (L3), a mass of γ neuron axons was labeled uniformly with cGFP driven by GAL4-201Y and with anti-FasII antibody. At 6 hr APF, however, many hole-like structures of unlabeled regions appeared in cross-sections. Considering that the number of varicosities was significantly reduced at this stage whereas most axons had not been disconnected, it is highly likely that the hole-like structures correspond to the vestiges of disappeared varicosities (Awasaki, 2004).

To confirm this, lobes labeled with the targeted expression of the presynapse marker n-Synaptobrevin::GFP (nSyb::GFP) and the membrane bound reporter mCD8::GFP (mGFP) were compared. Like cGFP, nSyb::GFP strongly labels varicosities along the axon branches. Images of the sections at 6 hr APF clearly reveal the hole-like structures. In contrast, mGFP labels voluminous varicosities much less intensively than cGFP, because these subcellular regions contain less membrane surface per volume. mGFP labels the hole-like structures much less clearly than does cGFP. This further supports the hypothesis that the unlabeled regions are associated with the disappearance of varicosities (Awasaki, 2004).

Each hole-like structure was considerably larger than the size of a single varicosity. The diameter of many holes was more than 5 μm, whereas the size of the largest varicosity was 1.8 μm. This indicates that each unlabeled hole corresponds to the vestige of multiple varicosities. Because the varicosities of a single neuron were scattered along its axon branch, it is unlikely that a single hole corresponds to the varicosities that belong to a single neuron. Rather, neighboring varicosities from different neurons must simultaneously disappear in a cluster (Awasaki, 2004).

The larval neuropil of the central nervous system of Drosophila is covered by sheaths of neuropil-associating glial cells. Three-dimensional reconstruction of the confocal serial sections revealed a smooth surface of the MB dorsal lobe in the larval brain. At 6 hr APF, however, the surface appeared eroded from the outside. This suggests that glial cells surrounding the MB lobe might erode this neuropil structure. The morphologic changes of glial cells were therefore traced by using the pan-glial driver GAL4-repo and the cGFP reporter (repo>cGFP). In larvae, glial cells surrounded the lobe, but the axon branches inside the lobe were devoid of glial processes. At 6 hr APF, glial processes had infiltrated into the lobes and formed many lumps. The positions of these lumps correspond to the hole-like structures that were unlabeled with anti-FasII antibody (Awasaki, 2004).

Close observation of the glial processes revealed various patterns of infiltration at 6 hr APF. In some sections, thin glial processes surround parts of axon branches labeled with anti-FasII antibody. Glial processes also spread into the surrounded mass, forming glial lumps. Vesicle-like structures in these glial lumps contained small dots that were FasII-positive. In the specimens with massive glial infiltration, glial processes formed large lumps that occupied whole regions unlabeled with anti-FasII antibody (Awasaki, 2004).

Considering that dorsal lobes are initially filled with FasII-positive axon mass and hole-like structures unlabeled with anti-FasII antibody are eventually filled with glial lumps labeled with repo>cGFP, images like the ones described are best interpreted as being intermediate steps. Hypothetically it would seem that at first, infiltrating glial processes surround clusters of varicosities, which are still labeled with anti-FasII antibody. Next, glial processes further penetrate between varicosities to form lumps. FasII-positive signals observed in the glial lumps might be parts of varicosities engulfed by glia. Intensity of the FasII signal within glia is remarkably reduced compared to the signals observed outside of glia. This might be because engulfed FasII protein is subject to intracellular degradation. Finally, the FasII signal totally disappears, and glial lumps replace the vestiges of varicosities (Awasaki, 2004).

If this hypothesis has validity, infiltrated parts of glia must exhibit evidence of high activity in the intracellular degradation process. To examine whether this is the case, vital staining was performed with LysoTracker, which labels acidic organelles such as late endosomes and lysosomes. In the larval lobes, no detectable signal was found with the LysoTracker staining. In the dorsal lobe at 6 hr APF, however, the LysoTracker-positive signals were constantly found. In most cases, the signals were localized in the lumps of the infiltrated glia. This finding strongly suggests that varicosities are engulfed by glia and degraded in the endosome-lysosome pathway (Awasaki, 2004).

Though most of the varicosities disappear by 12 hr APF, glial infiltration was still observed at 12 hr and 18 hr APF. Glial processes at these stages frequently form columnar structures lying along the axon branches. These columnar structures are often observed in the shaft region of the dorsal lobes. The disconnection of axons also occurs frequently in the shaft region rather than in the tip region at 12 hr APF. Thus, the formation of the columnar structures was spatially and temporally correlated with the occurrence of axon degeneration. This suggests that infiltrated glia are involved in the disconnection and degeneration of axon branches as well as the disappearance of varicosities (Awasaki, 2004).

These results so far suggested that varicosities and axon branches are engulfed and degraded in the glial lumps. There are two possibilities for the role of glia during this process: (1) axon branches of γ neurons might be degenerated by intrinsic mechanisms and infiltrating glia might just scavenge the already degraded debris; (2) degradation of the axon branches of the γ neurons may not be due to intrinsic mechanisms alone and may require infiltrating glia to actively break down the axon branches. To determine the most likely possibility, the pruning process was observed while glial cell function was conditionally inhibited by the targeted expression of the temperature-sensitive allele of shibire (shits1). The shibire gene is the Drosophila homolog of dynamin, which is involved in cellular membrane functions, including endocytosis, phagocytosis, and membrane growth. Because Shits1 protein acts as a dominant-negative at a restrictive temperature, targeted expression of Shits1 in glial cells would inhibit endocytosis and other membrane-related functions (Awasaki, 2004).

Larvae expressing Shits1 in glial cells by using the GAL4-repo driver (repo>shits1) were raised at a permissive temperature (19°C) and then placed in the restrictive temperature (29°C) from 0 hr APF to either 6 hr or 18 hr APF. Raising repo>shits1 pupae at the restrictive temperature results in an abnormal morphology of the glial cells. The most striking abnormality of these glia is that they did not extend their processes into the lobe at all. Whereas glial processes in a normal condition have infiltrated into lobes intensely already at 6 hr APF, dorsal lobes of repo>shits1 in the restrictive temperature were devoid of any glial processes even at 18 hr APF. This demonstrates that Shits1 almost completely suppresses the infiltration of glial processes (Awasaki, 2004).

Anti-FasII labeling of dorsal lobes revealed hole-like structures of unlabeled regions at 6 hr APF, and severe loss of axon branches at 18 hr APF. Neither holes of unlabeled regions nor loss of axon branches were observed in the lobes of the repo>shits1 animal in the restrictive temperature at 6 hr and 18 hr APF. The phenotypes were qualitatively indistinguishable among different individuals. These indicate that axon pruning including both disappearance of clustered varicosities and local axon degeneration were severely suppressed in these pupae (Awasaki, 2004).

Because inhibition of glial infiltration caused severe suppression of axon pruning, it is highly likely that infiltrating glia actively break down and remove axon branches rather than just scavenging already degraded debris. This result, together with the engulfment and degradation of clustered varicosities by glial lumps, suggests that infiltrating glia actively eliminate clustered varicosities at the early phase of pruning (Awasaki, 2004).

If the pruning of γ neurons is suppressed by the targeted disruption of glial function, do the unpruned axon branches remain in the adult MB? The repo>shits1 pupae incubated at the restrictive temperature did not survive until adult stage; therefore, glial function could not be disrupted throughout pupal development. When the temperature was shifted from the restrictive temperature to 25°C at 24 hr APF, however, a small number of survivors eclosed. In these animals (n = 10), abnormal axon branches expressing abnormal FasII-positive signals were observed shortly after hatching beside the α and β lobes, which are adult-specific branches labeled with anti-FasII antibody. Such abnormal FasII-positive branches were observed only in the distal tip of the lobes, because continuous fibers could not be traced from them to the shaft region of the lobe. Moreover, the abnormal branches were no longer visible in the brains more than 7 days after eclosion. It is therefore likely that these FasII-positive branches were the remnants of the γ neuron axons that were not pruned at 18 hr APF but gradually degenerated thereafter. Thus, transient inhibition of glial function in early pupae does not completely block axon pruning but severely delays the time course of axon pruning (Awasaki, 2004).

Axon pruning is triggered by a surge of ecdysone, which occurs just before puparium formation. The γ neurons express the ecdysone receptor EcR-B1 strongly in late third-instar larvae. The surrounding glial cells express EcR-B1 faintly at this stage. The expression of EcR-B1 in the glial cells, however, is unlikely to be associated with axon pruning. Although EcR-B mutation causes complete suppression of axon pruning, this suppression is rescued by expression of EcR-B1 only in the γ neurons. How, then, is the glial activation controlled? Because expression of EcR-B1 in γ neurons is sufficient for axon pruning, the glial cells must be activated via ecdysone stimulation of γ neurons. To test this hypothesis, expression of a dominant-negative form of the ecdysone receptor was induced in γ neurons by using the 201Y driver (201Y>EcR-DN), which drives expression in γ neurons, but not in the surrounding glia. If the activation of glia is dependent on the ecdysone-induced stimulation of γ neurons, glial infiltration would be suppressed in these pupae. Conversely, if the glial activation is independent of γ neurons, infiltration would not be disturbed. The results strongly suggest that ecdysone-induced stimulation of γ neurons is indispensable for the infiltrating and engulfing action of glial cells. Once γ neurons receive ecdysone by EcR-B1, these neurons would induce glial infiltration and engulfment. Thus, the neuron-glia interaction is essential for coordinated axon pruning (Awasaki, 2004).

The neuron-glia interactions for selective infiltration and engulfment resemble the interactions between phagocytes and apoptotic cells. It is not likely, however, that axon pruning and apoptosis use identical molecular mechanisms to activate glial cells and phagocytes. Mutation of Drosophila apoptosis activator genes (grim, hid, and rpr) and overexpression of apoptosis inhibitor genes (p35 and DIAP1) in γ neurons have no effect on axon pruning. However, Draper (Drpr), the homolog of the C. elegans cell corpse engulfment molecule CED-1, accumulates on the cell membrane of infiltrating glial processes. Engulfment of apoptotic neurons by glial cells is suppressed in the central nervous system (CNS) of drpr mutant embryos. This raises the possibility that sensing of apoptotic cells by phagocytes and sensing of unwanted axon branches by glial cells share a common molecular mechanism. Although the drpr mutant is isolated in Drosophila, it is embryonic lethal. Furthermore, clonal analysis with a flippase-mediated MARCM system does not work reliably in the larval and pupal glial cells. Analysis of the function of drpr in the engulfing glial cells with more sophisticated techniques to overcome the above problem, together with the analyses of the involvement of phosphatidylserine and lysophosphatidylcholine in the axon pruning of γ neurons, might reveal correlated mechanisms between removal of the cell corpse and unwanted axons (Awasaki, 2004).

Inhibition of glial function almost completely suppresses axon pruning until 18 hr APF, but the remaining axons still undergo degeneration in the adult. There is therefore a possibility that cell-autonomous mechanisms can prune axon branches without glial involvement. The time course of degeneration, however, is severely delayed (Awasaki, 2004).

Rapid degeneration of γ neuron axons seems vital for proper remodeling of the MB neural circuit. In the late larval brain, the α'/β' neurons extend their axons into the core of the axon bundle of γ neurons. As γ neuron axons are pruned during the early pupal stage, α'/β' neurons claim the region previously occupied by γ neurons. When pruning of γ neurons is suppressed by ectopic expression of yeast ubiquitin protease, UBP2, the unpruned axons remain in the vicinity of axon branches of α'/β' neurons in the adult. The morphology of α'/β' axon branches was distorted in this case. This suggests that proper elimination of larval axon branches might be important for the correct formation of adult axon branches. Rapid pruning with the help of the engulfing action of glia would be advantageous in such a situation (Awasaki, 2004).

Although there are many examples of axon pruning, the underlying mechanisms have not been identified except for Drosophila γ neurons and the mossy fiber pathway of the mouse hippocampal neurons. While axon pruning in Drosophila γ neurons is mediated by the degeneration of axons, which occurs within approximately 18 hr, the axon pruning of the mossy fiber pathway requires the retraction of axons over 500 μm in length, which occurs over more than 30 days. Further identification of the cellular and molecular mechanisms that mediate degeneration and retraction of axons might elucidate the differences and similarities in the strategy of each type of axon pruning (Awasaki, 2004).

Involvement of glia in motor neuron retraction during metamorphosis; Mediation by inputs from TGF-beta/BMP signaling and orphan nuclear receptors

Larval motor neurons remodel during Drosophila neuro-muscular junction dismantling at metamorphosis. This study describes the motor neuron retraction as opposed to degeneration based on the early disappearance of β-Spectrin and the continuing presence of Tubulin. By blocking cell dynamics with a dominant-negative form of Dynamin, this study shows that phagocytes have a key role in this process. Importantly, the presence of peripheral glial cells is shown close to the neuro-muscular junction that retracts before the motor neuron. In muscle, expression of EcR-B1 encoding the steroid hormone receptor required for postsynaptic dismantling, is under the control of the ftz-f1/Hr39 orphan nuclear receptor pathway but not the TGF-β signaling pathway. In the motor neuron, activation of EcR-B1 expression by the two parallel pathways (TGF-β signaling and nuclear receptor) triggers axon retraction. This study interrupted TGF-β signaling in motor neurons using expression of dominate negative Wishful thinking. It is proposed that a signal from a TGF-β family ligand is produced by the dismantling muscle (postsynapse compartment) and received by the motor neuron (presynaptic compartment) resulting in motor neuron retraction. The requirement of the two pathways in the motor neuron provides a molecular explanation for the instructive role of the postsynapse degradation on motor neuron retraction. This mechanism insures the temporality of the two processes and prevents motor neuron pruning before postsynaptic degradation (Boulanger, 2012).

It is a general feature of maturing brains, both in vertebrates and in invertebrates, that neural circuits are remodeled as the brain acquires new functions. In holometabolous insects, the difference in lifestyle is particularly apparent between the larval and the adult stages. These insects possess two distinct nervous systems at the larval and adult stages. A class of neurons is likely to function in both the larval and the adult nervous systems. The neuronal remodeling occurring during this developmental period is expected to be necessary for the normal functioning of the new circuits (Boulanger, 2012).

The pruning of an axon can involve a retraction of the axonal process, its degeneration or both a retraction and degeneration. The MB γ axon is pruned through a local degeneration mechanism. In contrast, axons may retract their cellular processes from distal to proximal in the absence of fragmentation and this mechanism is called retraction. Interestingly, the two mechanisms can occur sequentially in the same neuron, as in the case of the dendrites of the da neurons, where branches degenerate and the remnant distal tips retract (Boulanger, 2012).

This study provides evidence that the motor neuron innervating larval muscle 4 (NMJ 4) is pruned predominantly through a retraction mechanism. The first morphological indication of motor neuron retraction is the absence of fragmentation observed with anti-HRP staining at the level of the presynapse in all the developmental stages analyzed, together with a decrease in perimeter size observed after 2 h APF. The continuity of this HRP staining is in contrast to the pronounced interruptions between blebs observed with an antibody against mCD8 in γ axons. A molecular indication of motor neuron retraction in these studies is the fact that β-Spectrin disappears at the synapse 5 h APF, before motor neuron pruning takes place. Indeed, it has been shown using an RNA interference approach that loss of presynaptic β-Spectrin leads to presynaptic retraction and synapse elimination at the NMJ during larval stages. The modifications of the microtubule morphology that were observed, such as an increase in microtubule thickness and withdrawal, provide additional evidence of axonal retraction during NMJ remodeling. Finally, a strong argument in favor of a motor neuron retraction mechanism is the fact that Tubulin is present at the NMJ throughout all stages of axonal pruning at the start of metamorphosis (0-7 h APF). This stands in clear contrast to the abolition of Tubulin expression observed before the first signs of γ axon degeneration. It is also interesting to note that the motor neuron retraction observed in this study at metamorphosis and at larval stages are morphologically different. During metamorphosis, retraction bulbs or postsynaptic footprints, which have been reported at larval stages, were never visualized. The fact that the postsynapse dismantles at metamorphosis before motor neuron retraction might explain these discrepancies. Worth noting is the mechanistic correlation between accelerated debris shedding observed here for NMJ pruning at the start of metamorphosis and axosome shedding occurring during vertebrate motor neuron retraction (Boulanger, 2012).

In vertebrates, glia play an essential role in the developmental elimination of motor neurons. In Drosophila, the role of glia in sculpting the developing nervous system is becoming more apparent. Clear examples of a role for engulfing glial cells in axon pruning are well documented during the MB γ axon degeneration at metamorphosis. Also, glia are required for clearance of severed axons of the adult brain. A distinct protective role of glia has been recently discovered during the patterning of dorsal longitudinal muscles by motor neurobranches. This study describes the presence of glia processes close to the end of the pupal NMJ. The observations suggest that the glial extensions retract at 5 h APF, just before motor neuron retraction is observed. When the glial dynamic is blocked, the NMJ dismantling might be also blocked. It is hypothesized that during development in larvae and early pupae, glial processes have a protective role and aid in the maintenance of the NMJ. Then, between 2 and 5 h APF, glial retraction would be a necessary initial step that allows NMJ dismantling. In accordance with this hypothesis, glia play a protective role in the maintenance of NMJ during pruning of second order motor neuron branches 31 h APF (Boulanger, 2012).

Disruption of shi function specifically in glial cells results in an unpruned mushroom body γ neuron phenotype and prevents glial cell infiltration into the mushroom body (Awasaki, 2004). One can note that at the NMJ the role of the glia is proposed to be essentially opposite from its role in MB γ axons pruning but in both cases blocking the glia dynamics results in a similar blocking of the pruning process (Boulanger, 2012).

In vertebrates, phagocytes are recruited to the injured nerve where they clear, by engulfment, degenerating axons. In Drosophila, phagocytic blood cells engulf neuronal debris during elimination of da sensory neurons. This study shows that blocking phagocyte dynamics with shi produces a strong blockade of the NMJ dismantling process. One possibility is that phagocytes attack and phagocytose the postsynaptic material, a process blocked by compromising shi function resulting in postsynaptic protection. In accordance, it has been shown that phagocytes attack not only the da dendrites to be pruned, but also the epidermal cells that are the substrate of these dendrites (Boulanger, 2012).

During NMJ dismantling, the muscle has an instructive role for motor neuron retraction. In all the situations where postsynapse dismantling is blocked, the corresponding presynaptic motor neuron retraction is also blocked. Therefore, it is sufficient to propose that both glial cells and phagocytes affect only the postsynaptic compartment. Nevertheless, one cannot rule out that these two cell types both act directly at the pre and at the postsynapse (Boulanger, 2012).

ECR-B1 is highly expressed and/or required for pruning in remodeling neurons of the CNS. MB γ neurons and antennal lobe projection neurons remodeling require both the same TGF-β signaling to upregulate EcR-B1. In the MBs only neurons destined to remodel show an upregulation of EcR-B1. At least two independent pathways insure EcR-B1 differential expression. The TGF-β pathway and the nuclear receptor pathway are thought to provide the necessary cell specificity of EcR-B1 transcriptional activation. This study shows that in the motor neuron pruning these two pathways are also necessary to activate EcR-B1. Noteworthy, showing an analogous requirement of ftz-f1/Hr39 pathway in two different remodeling neuronal systems unravels the fundamental importance of this newly described pathway (Boulanger, 2012).

The following model is proposed for the sequential events that are occurring during NMJ dismantling at early metamorphosis. First, EcR-B1 is expressed in the muscle under the control of FTZ-F1. FTZ-F1 activates EcR-B1 and represses Hr39. This repression is compulsory for EcR-B1 activation. Importantly, TGF-β/BMP signaling does not appear to be required for EcR-B1 activation in this tissue, however, a result of EcR-B1 activation in the muscle would be the production of a secreted TGF-β family ligand. Then, this secreted TGF-β family ligand reaches the appropriate receptors and activates the TGF-β signaling in the motor neuron. Finally, TGF-β signaling in association with the nuclear receptor pathway activates EcR-B1 expression resulting in motor neuron retraction. Since glial cells and phagocytes are required for the dismantling process, it is possible that a TGF-β/BMP family ligand(s) be produced by one or both of these cell types and not by the postsynaptic compartment. Noteworthy, a recent study shows that glia secrete myoglianin, a TGF-β ligand, to instruct developmental neural remodeling in Drosophila MBs (Awasaki, 2011). Nevertheless, one can note that the requirement of the two pathways in the motor neuron provides a simple molecular explanation of the instructive role of postsynapse degradation on motor neuron retraction. This mechanism insures the temporality of the two processes and prevents motor neuron pruning before postsynaptic degradation. It was proposed that in the MBs, the association of these two pathways provides the cell (spatial) specificity of pruning. In this paper, this association is proposed to provide the temporal specificity of the events. Future studies will be necessary to understand how EcR-B1 controls the production of a TGF-β/BMP ligand(s) in the muscle, the reception of this signal by the motor neuron and the ultimate response by the motor neuron to initiate retraction. These steps will be necessary to unravel the molecular mechanisms underlying the NMJ dismantling process and related phenomenon in vertebrate NMJ development and disease. Interestingly, it appears that TGF-β ligands on the one hand are positive regulators of synaptic growth during larval development and on the other hand, they are positive regulators of synaptic retraction, at the onset of metamorphosis. In both situations signaling provides a permissive role, sending a signal from the target tissue to the neuron. The consequence of this signal would be dependent on developmental timing thus, on a change in context (Boulanger, 2012).

Astrocytes engage unique molecular programs to engulf pruned neuronal debris from distinct subsets of neurons

Precise neural circuit assembly is achieved by initial overproduction of neurons and synapses, followed by refinement through elimination of exuberant neurons and synapses. Glial cells are the primary cells responsible for clearing neuronal debris, but the cellular and molecular basis of glial pruning is poorly defined. This study shows that Drosophila larval astrocytes transform into phagocytes through activation of a cell-autonomous, steroid-dependent program at the initiation of metamorphosis and are the primary phagocytic cell type in the pupal neuropil. The developmental elimination was examined of two neuron populations-mushroom body (MB) gamma neurons and vCrz+ neurons (expressing Corazonin [Crz] neuropeptide in the ventral nerve cord [VNC])-where only neurites are pruned or entire cells are eliminated, respectively. MB gamma axons were found to be engulfed by astrocytes using the Draper and Crk/Mbc/dCed-12 signaling pathways in a partially redundant manner. In contrast, while elimination of vCrz+ cell bodies requires Draper, elimination of vCrz+ neurites is mediated by Crk/Mbc/dCed-12 but not Draper. Intriguingly, it was also found that elimination of Draper delayed vCrz+ neurite degeneration, suggesting that glia promote neurite destruction through engulfment signaling. This study identifies a novel role for astrocytes in the clearance of synaptic and neuronal debris and for Crk/Mbc/dCed-12 as a new glial pathway mediating pruning and reveals, unexpectedly, that the engulfment signaling pathways engaged by glia depend on whether neuronal debris was generated through cell death or local pruning (Tasdemir-Yilmaz, 2013).

Astrocytes serve as regulators of synapse formation and function and are generally supportive of neural circuits. Based on several lines of evidence, this study showed that astrocytes transform morphologically and functionally into phagocytes at pupariation and engulf significant amounts of neural debris. Phagocytic astrocytes take on a highly vacuolated appearance, up-regulate the engulfment molecule Draper, contain cytoplasmic vacuoles filled with debris that stains for synaptic or axonal markers, and exhibit high levels of lysosomal activity. Activation of this phagocytic program depends on cell-autonomous signaling through the EcR, since blockade of EcR in even single astrocytes suppressed their transformation by all morphological and molecular criteria. Blockade of astrocyte phagocytic function by multiple methods (e.g., EcRDN or Shits) suppressed the clearance of synapses throughout the CNS, axons of MB γ neurons in the central brain, and the neurites of vCrz+ cells in the VNC. This latter observation defines vCrz+ neurons as a new system to explore astrocyte-neuron interactions during neuronal apoptosis and neurite or synapse elimination in the CNS (Tasdemir-Yilmaz, 2013).

While roles for Drosophila glia in the engulfment of pruned MB γ axons have been previously described, it was unexpected that astrocytes would be the phagocytic cell type. In the adult Drosophila brain, a second type of neuropil glial cells, ensheathing glia, is responsible for engulfing injured axonal debris, while astrocytes fail to respond in a detectable way morphologically or molecularly (e.g., by Draper up-regulation). The stark difference in glial subtypes executing phagocytic function in the pupa versus the adult could result from differences in glial genetic programs during development versus in mature glia or indicate a key difference in the molecular nature of pruned neurites compared with those undergoing injury-induced axon degeneration (Tasdemir-Yilmaz, 2013).

The extent to which mammalian astrocytes are phagocytes in vivo has remained unclear until recently. In culture, astrocytes can be phagocytic and engulf apoptotic cells or amyloid-β. In vivo, the post-laminar optic nerve head myelination transition zone (MTZ) astrocytes express Mac-2, a molecule implicated in phagocytic activity, and internalize axonal evulsions, and this event appears to be increased in frequency in glaucoma models. Most impressively, it has been recently demonstrated that astrocytes engulf synaptic material in vivo and that this event is mediated by MEGF10 (mouse Draper) and the engulfment receptor MERTK. Thus, Draper/MEGF10-dependent engulfment of pruned synapses appears to be an ancient astrocytic mechanism; whether the same is true for the Crk/Mbc/dCed-12 pathway remains to be determined. Transcriptome data from purified mouse forebrain astrocytes support this notion, since molecular pathways for engulfment are highly enriched, including, in addition to MEGF10, Gulp1 (dCed-6), and in addition to Crk, Dock1 (Mbc) and Elmo (dCed-12). It will be important to explore whether these additional pathways have similar roles in astrocytes for the pruning of mammalian neural circuits during development and whether astrocyte engulfment activity is modified in neurological diseases involving axonal, dendritic, or synaptic loss (Tasdemir-Yilmaz, 2013).

Two types of neurons that undergo different types of developmental reorganization were examined. MB γ neurons prune their medial and dorsal axon branches and dendrites, their cell bodies remain viable, and, at midpupal stages, they re-extend medial axon branches to establish adult-specific connectivity. In contrast, vCrz+ neurons exhibit complete neurite degeneration, and cell bodies undergo apoptotic death and eventually are completely eliminated. In addition to there being remarkable differences in the patterns of fragmentation exhibited by these subsets of neurons, there are also critical differences in the engulfment signaling pathways used to promote their initial destruction and clearance (Tasdemir-Yilmaz, 2013).

Previous work has revealed a role for Draper in MB γ neuron pruning, with draper-null mutants exhibiting a delay of ~2 d in the clearance of pruned MB γ neuron axonal debris. This study provides evidence supporting a key role for the Crk/Mbc/dCed-12 complex in the clearance of MB γ neuron axonal debris and demonstrates that Crk/Mbc/dCed-12 knockdown and draper mutants have additive phenotypes, indicating that these signaling pathways act in a partially redundant fashion in astrocytes to promote clearance of pruned MB γ neuron axons. While these phenotypes are additive, it is noted that draper mutants exhibit a stronger delay in MB γ neuron pruning, suggesting that Draper signaling plays a more prominent role in this brain region than signaling through the Crk/Mbc/dCed-12 complex. However, the possibility that a Crk, mbc, or dCed-12 mutant might have a stronger phenotype than the RNAi lines used in this study cannot be excluded (Tasdemir-Yilmaz, 2013).

vCrz+ neuronal clearance appears to involve both Draper signaling and the Crk/Mbc/dCed-12 complex, with the former primarily promoting clearance of vCrz+ cell bodies, and the latter driving clearance of degenerating vCrz+ neurites. In draper-null mutants at 18 h APF, the majority of neuronal cell bodies remained at the edge of the neuropil, while neurite debris was largely cleared. Reciprocally, astrocytic knockdown of dCed-12 suppressed neurite clearance, while vCrz+ cell bodies were promptly eliminated. The lack of additivity of the phenotypes for either neurite or cell body clearance in draper-null mutants with astrocytic dCed-12RNAi suggests that elimination of cell bodies is primarily driven by the Draper pathway, while neurite clearance is largely accomplished by signaling through the Crk/Mbc/dCed-12 complex (Tasdemir-Yilmaz, 2013).

This study reveals that the genetic pathways engaged by glia to engulf pruned neuronal material versus apoptotic neurons are context-dependent and correlate with the type of destructive program. Interestingly, the molecular pathways that mediate axonal degeneration during axon pruning versus apoptosis are also distinct in mammalian cultured neurons. Local withdrawal of NGF in the axon activates Caspase 6-dependent axon degeneration, which is not sensitive to XIAP1 inhibition. In contrast, whole-cell NGF withdrawal leads to Caspase 6-independent apoptotic cell death and degeneration of axons, which is then sensitive to XIAP1. These observations argue that context matters when neurites and cell bodies are being destroyed. It is speculated that neuron-glia signaling during engulfment events might also be compartmentalized, with neurites and cell bodies generating different types of 'eat me' cues for clearance by glia (Tasdemir-Yilmaz, 2013).

Do engulfing glial cells actively promote the destruction of target neurons? The intriguing observation was made that loss of a single copy of draper is sufficient to dominantly suppress the elimination of vCrz+ cell bodies and neurites at 6 h APF. Moreover, loss of two copies results in the retention of nearly all vCrz+ cell bodies and significant parts of the vCrz+ scaffold, and many regions of the scaffold appeared morphologically intact, suggesting a delay of neurite fragmentation. Previous work has shown that expression of the anti-apoptotic molecule P35 is sufficient to suppress the pruning of vCrz+ neurites for at least 1 d; this study extended this observation and found this to be true for at least 2 d . Thus, blocking apoptosis in vCrz+ is sufficient to significantly delay neurite degeneration. This observation, coupled with the known role for CED-1 in actively promoting the death of engulfment targets, suggests that engulfing nonastrocyte glia may promote neuronal apoptosis through Draper signaling. This in turn would promote degeneration of the neurite scaffold after cell body death. If this underlies the delay that was observed in neurite degeneration in draper mutants, it would argue that glia actively sculpt neural networks by promoting the destruction of selected subsets of neurons. Subsequent work exploring the survival of cell bodies preserved in draper-null animals will be essential to explore this exciting possibility (Tasdemir-Yilmaz, 2013).

Astrocytes play a key role in Drosophila mushroom body axon pruning

Axon pruning is an evolutionarily conserved strategy used to remodel neuronal connections during development. The Drosophila mushroom body (MB) undergoes neuronal remodeling in a highly stereotypical and tightly regulated manner, however many open questions remain. Although it has been previously shown that glia instruct pruning by secreting a TGF-beta ligand, myoglianin, which primes MB neurons for fragmentation and also later engulf the axonal debris once fragmentation has been completed, which glia subtypes participate in these processes as well as the molecular details are unknown. This study shows that, unexpectedly, astrocytes are the major glial subtype that is responsible for the clearance of MB axon debris following fragmentation, even though they represent only a minority of glia in the MB area during remodeling. Furthermore, astrocytes both promote fragmentation of MB axons as well as clear axonal debris and that this process is mediated by ecdysone signaling in the astrocytes themselves. In addition, this study found that blocking the expression of the cell engulfment receptor Draper in astrocytes affects only axonal debris clearance. Thereby this study uncoupled the function of astrocytes in promoting axon fragmentation to that of clearing axonal debris after fragmentation has been completed. This study finds a novel role for astrocytes in the MB and suggests two separate pathways in which they affect developmental axon pruning (Hakim 2014).

DRK/DOS/SOS converge with Crk/Mbc/dCed-12 to activate Rac1 during glial engulfment of axonal debris

Nervous system injury or disease leads to activation of glia, which govern postinjury responses in the nervous system. Axonal injury in Drosophila results in transcriptional upregulation of the glial engulfment receptor Draper; there is extension of glial membranes to the injury site (termed activation), and then axonal debris is internalized and degraded. Loss of the small GTPase Rac1 from glia completely suppresses glial responses to injury, but upstream activators remain poorly defined. Loss of the Rac guanine nucleotide exchange factor (GEF) Crk/myoblast city (Mbc)/dCed-12 has no effect on glial activation, but blocks internalization and degradation of debris. This study shows that the signaling molecules Downstream of receptor kinase (DRK) and Daughter of sevenless (DOS) (mammalian homologs, Grb2 and Gab2, respectively) and the GEF Son of sevenless (SOS) (mammalian homolog, mSOS) are required for efficient activation of glia after axotomy and internalization/degradation of axonal debris. At the earliest steps of glial activation, DRK/DOS/SOS function in a partially redundant manner with Crk/Mbc/dCed-12, with blockade of both complexes strongly suppressing all glial responses, similar to loss of Rac1. This work identifies DRK/DOS/SOS as the upstream Rac GEF complex required for glial responses to axonal injury, and demonstrates a critical requirement for multiple GEFs in efficient glial activation after injury and internalization/degradation of axonal debris (Lu, 2014).

Astrocytic glutamate transport regulates a Drosophila CNS synapse that lacks astrocyte ensheathment

Anatomical, molecular, and physiological interactions between astrocytes and neuronal synapses regulate information processing in the brain. The fruit fly Drosophila melanogaster has become a valuable experimental system for genetic manipulation of the nervous system and has enormous potential for elucidating mechanisms that mediate neuron-glia interactions. This study shows the first electrophysiological recordings from Drosophila astrocytes and characterizes their spatial and physiological relationship with particular synapses. Astrocyte intrinsic properties were found to be strongly analogous to those of vertebrate astrocytes, including a passive current-voltage relationship, low membrane resistance, high capacitance, and dye-coupling to local astrocytes. Responses to optogenetic stimulation of glutamatergic pre-motor neurons were correlated directly with anatomy using serial electron microscopy reconstructions of homologous identified neurons and surrounding astrocytic processes. Robust bidirectional communication was present: neuronal activation triggered astrocytic glutamate transport via Eaat1, and blocking Eaat1 extended glutamatergic interneuron-evoked inhibitory post-synaptic currents in motor neurons. The neuronal synapses were always located within a micron of an astrocytic process, but none were ensheathed by those processes. Thus, fly astrocytes can modulate fast synaptic transmission via neurotransmitter transport within these anatomical parameters (MacNamee, 2016).

The astrocyte network in the ventral nerve cord neuropil of the Drosophila third-instar larva

Understanding neuronal function at the local and circuit level requires understanding astrocyte function. This study provides a detailed analysis of astrocyte morphology and territory in the Drosophila third-instar ventral nerve cord where there already exists considerable understanding of the neuronal network. Astrocyte shape varies more than previously reported; many have bilaterally symmetrical partners, many have a high percentage of their arborization in adjacent segments, and many have branches that follow structural features. Taken together, these data are consistent with, but not fully explained by, a model of a developmental growth process dominated by competitive or repulsive interactions between astrocytes. The data suggest that the model should also include cell-autonomous aspects, as well as the use of structural features for growth. Variation in location of arborization territory for identified astrocytes was great enough that a standardized scheme of neuropil division among the six astrocytes that populate each hemi-segment is not possible at the third instar. The arborizations of the astrocytes can extend across neuronal functional domains. The ventral astrocyte in particular, whose territory can extend well into the proprioceptive region of the neuropil, has no obvious branching pattern that correlates with domains of particular sensory modalities, suggesting that the astrocyte would respond to neuronal activity in any of the sensory modalities, perhaps integrating across them. This study sets the stage for future studies that will generate a robust, functionally oriented connectome that includes both partners in neuronal circuits-the neurons and the glial cells, providing the foundation necessary for studies to elucidate neuron-glia interactions in this neuropil (Hernandez, 2020).

Remodeling of peripheral nerve ensheathment during the larval-to-adult transition in Drosophila

Over the course of a four-day period of metamorphosis, the Drosophila larval nervous system is remodeled. In peripheral nerves in the abdomen, five pairs of abdominal nerves (A4-A8) fuse to form the terminal nerve trunk. This reorganization is associated with selective remodeling of four layers that ensheath each peripheral nerve. The neural lamella (NL), is the first to dismantle; its breakdown is initiated by 6 hours after puparium formation, and is completely removed by the end of the first day. This layer begins to re-appear on the third day of metamorphosis. Perineurial Glial (PG) cells situated just underneath the NL, undergo significant proliferation on the first day of metamorphosis, and at that stage contribute to 95% of the glial cell population. Cells of the two inner layers, Sub-Perineurial Glia (SPG) and Wrapping Glia (WG) increase in number on the second half of metamorphosis. Induction of cell death in perineurial glia via the cell death gene reaper and the Diptheria toxin (DT-1) gene, results in abnormal bundling of the peripheral nerves, suggesting that perineurial glial cells play a role in the process. A significant number of animals fail to eclose in both reaper and DT-1 targeted animals, suggesting that disruption of PG also impacts eclosion behavior. These studies will help establish the groundwork for further work on cellular and molecular processes that underlie the co-ordinated remodeling of glia and the peripheral nerves they ensheath (Subramanian, 2017).

Loss- or gain-of-function mutations in ACOX1 cause axonal loss via different Mechanisms

ACOX1 (acyl-CoA oxidase 1) encodes the first and rate-limiting enzyme of the very-long-chain fatty acid (VLCFA) beta-oxidation pathway in peroxisomes and leads to H2O2 production. Unexpectedly, Drosophila ACOX1 is mostly expressed and required in glia, and loss of ACOX1 leads to developmental delay, pupal death, reduced lifespan, impaired synaptic transmission, and glial and axonal loss. Patients who carry a previously unidentified, de novo, dominant variant in ACOX1 (p.N237S) also exhibit glial loss. However, this mutation causes increased levels of ACOX1 protein and function resulting in elevated levels of reactive oxygen species in glia in flies and murine Schwann cells. ACOX1 (p.N237S) patients exhibit a severe loss of Schwann cells and neurons. However, treatment of flies and primary Schwann cells with an antioxidant suppressed the p.N237S-induced neurodegeneration. In summary, both loss and gain of ACOX1 lead to glial and neuronal loss, but different mechanisms are at play and require different treatments (Chung, 2020).

Lipids are critical for neuronal development, synaptic plasticity, and function. Abnormal lipid metabolism contributes to the pathogenesis of several neurodegenerative disorders, including Alzheimer's disease, Parkinson's disease, and various diseases associated with glial dysfunction. Mitochondria and peroxisomes have been implicated in some of these neurodegenerative diseases, but the molecular events that underlie the demise of neurons vary widely. Some neurodegenerative diseases have been associated with defects in the degradation of fatty acids by β-oxidation, and these disorders share some common features. In yeast and plants, peroxisomes are the only site of β-oxidation. However, in vertebrates and insects, peroxisomes have more restricted functions as they are the site of β-oxidation of VLCFA, whereas long and medium chain fatty acids are processed in mitochondria (Chung, 2020).

Peroxisomal ACOX1 (acyl‐CoA oxidase 1) is the first and rate‐limiting enzyme in fatty acid β‐oxidation of VLCFA and a major producer of hydrogen peroxide (H2O2). ACOX1 deficiency (OMIM #264470) in humans is an autosomal recessive disorder that causes a rapid and severe loss of nervous system function. Newborns with ACOX1 deficiency exhibit hypotonia and seizures, then they experience a gradual loss of learning and speaking skills, usually beginning between the ages of 1 and 3. As the condition worsens, children develop exaggerated reflexes, have an increase in VLCFA, increased muscle tone, severe and recurrent seizures, and loss of vision and hearing. Most children with peroxisomal ACOX1 deficiency do not survive past early childhood (Chung, 2020).

Previous work has shown that loss of various peroxisomal proteins and enzymes causes severe neuronal defects in flies (Di Cara, 2019, Mast, 2011, Wangler, 2017). Exactly how loss of peroxisomes contributes to severe neurologic phenotypes in flies, mice, and humans is still poorly explored. This study reports that dACOX1 is expressed in glia and that loss of dACOX1 severely affects lifespan, increases VLCFA, causes loss of vision, and leads to glial loss and reduced neuronal survival in flies. In contrast, a novel variant, p.N237S, identified in three patients with progressive ataxia and hearing loss, stabilizes ACOX1 as an active dimer, acts as a gain-of-function mutation, produces elevated levels of reactive oxygen species (ROS) in insulating glia, does not alter VLCFA levels, and leads to the demise of wrapping glia in flies and primary Schwann cells in mouse. A potent antioxidant, N-acetyl cysteine amide (NACA), strongly suppresses these effects. These data show that proper activity of peroxisomal β-oxidation is essential for glial survival, and both loss and gain of ACOX1 severely affect glial function in flies and humans, albeit via different pathways (Chung, 2020).

Flies have no myelin, and this has often been raised as a concern for modeling glial disease in flies. However, the current data indicate striking parallels between wrapping glia and Schwann cells. Myelin is rich in VLCFA and ELOVL1, an enzyme that synthesizes VLCFA from LCFA, and VLCFA are required for myelin maintenance. Indeed, VLCFA are incorporated in glycerophospholipids and most VLCFAs in mammals are present in sphingolipids, lipids that are abundant in the outer leaflet of the plasma membrane, especially in lipid rafts and myelin where they reduce the fluidity of membranes. The ratio of C24 sphingolipids/total sphingolipids varies in different tissues. In mice, C24 sphingomyelin accounts for 25% of the sphingomyelin in the brain, and VLCFA are converted to C24 sphingolipids by ceramide synthase 2 (CerS2), which is required for myelin maintenance. Loss of CerS2 leads to an 80% reduction of myelin basic protein. Hence, VLCFAs are critical for myelin maintenance (Chung, 2020).

Interestingly, a form of the progressive neuropathy Charcot-Marie-Tooth disease (CMT1A) is caused by a mutation in PMP2, which encodes Fatty Acid Binding Protein 8 that is abundantly expressed in Schwann cells and binds LCFA. Glia in flies do not contain myelin, but the wrapping glia that insulate axons are rich in the ceramide phosphoethanolamine (CPE), a lipid that is very similar to sphingomyelin. Depletion of CPE results in defasciculation and failure of wrapping glia to enwrap peripheral axons reminiscent of defasciculation observed in CMT. Previous work has shown that VLCFAs are present in CPE in flies (Lin, 2018); based on these data it is estimated that 67% of all CPEs contain VLCFAs above C22 in length and 19% contain FA longer than C24. The data indicate that an increase in VLCFA upon loss of ACOX1 also causes a defasciculation and failure of wrapping glia to ensheath axons. Similarly, patients with loss of ACOX1 also exhibit demyelinating peripheral and central neuropathies. Hence, not being able to break down VLCFA is toxic and most likely severely affects the membrane composition of wrapping glia and myelin, drawing striking parallels between myelin in vertebrate glia and CPE in wrapping glia in insects. The current findings that bezafibrate, a drug that inhibits the synthesis of VLCFA, strongly suppresses the phenotypes in ACOX1-deficient flies clearly supports the evidence that elevated levels of VLCFA in glia may be a key trigger for glial and axonal loss (Chung, 2020).

The data show that ACOX1 is abundant in glia and poorly expressed in neurons in both flies and mice. This finding is in agreement with previous observations in vertebrates that peroxisomes are abundant in Schwann cells, but absent in the axons they enwrap. The breakdown of myelin leads to the production of VLCFAs, which, through β-oxidation in peroxisomes, produce LCFA that can be used as an energy source in the mitochondria of glial cells. Glial cells are known to provide lactate to neurons as an energy source in vertebrates. Kassmann (2014) proposed that LCFA produced in glia may correspond to a second energy source for axons. Previous work has shown that lipids can be transferred from neurons to glia, requiring fatty acid transport protein and apolipoprotein D (Dourlen, 2012, Liu, 2017, Liu, 2015). A similar export pathway may transport fatty acids from glia to neurons (Kis, 2015). Hence, the reduction in LCFA in glia upon loss of ACOX1 may also be detrimental for axons and deprive them of an energy source (Chung, 2020).

In mammals, peroxisomes are observed in astrocytes, oligodendrocytes, microglia, and Schwann cells. Although mice that lack the peroxisomal proteins Pex5 or Pex11 display severe neurological defects, removal of Pex5 specifically from neurons or from astrocytes does not cause obvious phenotypes. In contrast, removal of Pex5 from oligodendrocytes (CNP-Pex5 knockout) induces demyelination and severe axonal loss, suggesting that axonal loss is caused by peroxisome-deficient oligodendrocytes. In addition, mice that lack Mfp2, a VLCFA metabolizing enzyme that acts downstream of Acox1, also exhibit severe loss of axons. Again, these defects are not observed when Mfp2 is only absent from neurons or astrocytes. However, mice deficient for Mfp2 in oligodendrocytes do not show prominent pathology in the first year of life. This is inconsistent with observations in flies and humans, suggesting some redundancy for Mfp2 in mice. Finally, loss of Acox1 in mice has not been associated with axonal loss in the first 6 months of life, nor has a neurodegenerative phenotype been documented yet. Hence, humans and flies share similar phenotypes in the absence of ACOX1 that have not yet been observed in mice. It is proposed that this may be due to redundancy with murine Acox2 and Acox3 (Chung, 2020).

Monitoring cell-cell contacts in vivo in transgenic animals

This study used a synthetic genetic system based on ligand-induced intramembrane proteolysis to monitor cell-cell contacts in animals. Upon ligand-receptor interaction in sites of cell-cell contact, the transmembrane domain of an engineered receptor is cleaved by intramembrane proteolysis and releases a protein fragment that regulates transcription in the interacting partners. The system can be used to regulate gene expression between interacting cells, both in vitro and in vivo, in transgenic Drosophila. The system allows for detection of interactions between neurons and glia in the Drosophila nervous system. In addition, it was observed that when the ligand is expressed in subsets of neurons with a restricted localization in the brain it leads to activation of transcription in a selected set of glial cells that interact with those neurons. This system will be useful to monitor cell-cell interactions in animals, and can be used to genetically manipulate cells that interact with one another (Huang, 2016).

Mactosylceramide prevents glial cell overgrowth by inhibiting insulin and fibroblast growth factor receptor signaling

Receptor Tyrosine Kinase (RTK) signaling controls key aspects of cellular differentiation, proliferation, survival, metabolism, and migration. Deregulated RTK signaling also underlies many cancers. Glycosphingolipids (GSL) are essential elements of the plasma membrane. By affecting clustering and activity of membrane receptors, GSL modulate signal transduction, including that mediated by the RTK. GSL are abundant in the nervous system, and glial development in Drosophila is emerging as a useful model for studying how GSL modulate RTK signaling. Drosophila has a simple GSL biosynthetic pathway, in which the mannosyltransferase Egghead controls conversion of glucosylceramide (GlcCer) to mactosylceramide (MacCer). Lack of elongated GSL in egghead (egh) mutants causes overgrowth of subperineurial glia (SPG), largely due to aberrant activation of phosphatidylinositol 3-kinase (PI3K). However, to what extent this effect involves changes in upstream signaling events is unresolved. This study shows that glial overgrowth in egh is strongly linked to increased activation of insulin and Fibroblast Growth Factor receptor (FGFR). Glial hypertrophy is phenocopied when overexpressing gain-of-function mutants of the Drosophila Insulin Receptor (InR) and the FGFR homolog Heartless (Htl) in wild type SPG, and is suppressed by inhibiting Htl and InR activity in egh. Knockdown of GlcCer synthase in the SPG fails to suppress glial overgrowth in egh nerves, and slightly promotes overgrowth in wild type, suggesting that RTK hyperactivation is caused by absence of MacCer and not by GlcCer accumulation. It is concluded that an early product in GSL biosynthesis, MacCer, prevents inappropriate activation of Insulin and Fibroblast Growth Factor Receptors in Drosophila glia (Gerdoe-Kristensen, 2016).

TRAP-seq profiling and RNAi-based genetic screens identify conserved glial genes required for adult Drosophila behavior

Although, glial cells have well characterized functions in the developing and mature brain, it is only in the past decade that roles for these cells in behavior and plasticity have been delineated. Glial astrocytes and glia-neuron signaling, for example, are now known to have important modulatory functions in sleep, circadian behavior, memory and plasticity. To better understand mechanisms of glia-neuron signaling in the context of behavior, cell-specific, genome-wide expression profiling was conducted of adult Drosophila astrocyte-like brain cells and RNA interference (RNAi)-based genetic screens were performed to identify glial factors that regulate behavior. Importantly, these studies demonstrate that adult fly astrocyte-like cells and mouse astrocytes have similar molecular signatures; in contrast, fly astrocytes and surface glia-different classes of glial cells-have distinct expression profiles. Glial-specific expression of 653 RNAi constructs targeting 318 genes identified multiple factors associated with altered locomotor activity, circadian rhythmicity and/or responses to mechanical stress (bang sensitivity). Of interest, one of the relevant genes encodes a vesicle recycling factor, four encode secreted proteins and three encode membrane transporters. These results strongly support the idea that glia-neuron communication is vital for adult behavior (Ng, 2016).

Dynamic analysis of the mesenchymal-epithelial transition of blood-brain barrier forming glia in Drosophila

During development, many epithelia are formed by a mesenchymal-epithelial transition (MET). This study examined the major stages and underlying mechanisms of MET during blood-brain barrier (BBB) formation in Drosophila. Contact with the basal lamina was shown to be essential for the growth of the barrier-forming subperineurial glia (SPG). Septate junctions (SJs), which provide insulation of the paracellular space, are not required for MET, but are necessary for the establishment of polarized SPG membrane compartments. In vivo time-lapse imaging reveals that the Moody GPCR signalling pathway regulates SPG cell growth and shape, with different levels of signalling causing distinct phenotypes. Timely, well-coordinated SPG growth is essential for the uniform insertion of SJs and thus the insulating function of the barrier. This is the first dynamic in vivo analysis of all stages in the formation of a secondary epithelium and of the key role trimeric G protein signalling plays in this important morphogenetic process (Schwabe, 2017).

This study of Drosophila BBB development represents the first dynamic in vivo study of MET and secondary epithelium formation. The data shed particular light on the roles of the basal lamina and of the insulating SJs, as well as on the function of GPCR signaling in this important morphogenetic process. Once SPG reach the CNS surface, contact with the basal lamina is essential for the extensive growth of the SPG during epithelium formation. Previous in vitro studies have shown that adhesion to basal lamina components is necessary for cell spreading and proliferation, however this study is the first to demonstrate in vivo that attachment to the basal lamina is essential for non-proliferative cell growth and ensheathment. Attachment to the ECM occurs primarily through focal adhesions and integrins, which in turn can activate MAPK signaling, triggering cell proliferation and growth. In addition, adhesion to the ECM has been shown to provide traction, which facilitates cell spreading. Contact to the ECM may thus provide the SPG with both growth signals and attachment sites. Being highly expressed on the basal lamina facing side of SPG, Dystroglycan (Dg) is an excellent candidate for mediating ECM attachment. However, zygotic mutants of Dg show no BBB defects and germline clones could not be analyzed due to Dg's role in oogenesis (Schwabe, 2017).

Beyond supporting SPG growth, contact with the basal lamina likely provides an important cue for polarizing the cells, as judged by their strong enrichment of Dg at the basal lamina facing (basal) membrane compartment. Previous studies have shown that Dg and its ligand Pcan/Trol are required for the establishment of polarity in follicle cells. However, when the basal lamina and thus its ligand Pcan are depleted, Dg is still expressed and polarized in the SPG, suggesting that glial polarity can be supported by the residual basal lamina or that additional polarizing signals exist (Schwabe, 2017).

Once SJs have formed, the GPCR Moody and the Mdr65 transporter are asymmetrically distributed within the SPG, further demonstrating that these cells possess distinct apical and basal membrane compartments. This polarized distribution is coincident with and dependent on the presence of SJs, demonstrating for the first time that SJs serve a function in cell polarity. By acting as a fence and preventing diffusion of membrane proteins across the lateral compartment, the SJs maintain asymmetric protein distributions, which could result from polarized exocytosis or endocytosis. Intriguingly, a separate study has identified PKA as a crucial antagonistic effector of Moody signaling. PKA has been shown to regulate polarized exocytosis at the trans-Golgi network in different types of epithelia. Apical-basal polarity plays an important morphogenetic role in the continued growth of the SPG epithelium during larval stages and in the function of the BBB (Schwabe, 2017).

Signaling by the GPCR Moody plays a critical role both in regulating the growth of individual SPG and in synchronizing this process across the entire SPG cell population. In Moody pathway mutants, glial growth behavior is more erratic, and more variable between cells. This increased variability of glial cell shape, size, and growth causes a significant delay of epithelial closure of up to 1.5 hrs. This delay is not caused by an earlier delay in glial migration or by a delay in SJ formation (Schwabe, 2017).

The detailed dynamic analysis reveals that, in moody and loco mutants, the spatio-temporal coordination of cell spreading is impaired. Spreading cells, like other motile cells, show fluctuating exploratory motions of the leading edge visible as cycles of protrusion and retraction. This complex process can be broken down into discrete steps: actin protrusion of the leading edge, adhesion to the ECM, and myosin-driven contraction against adhesions. Time-lapse recordings indicate that Moody signaling has its most pronounced effect on the stabilization of protrusions, as evidenced by an increase in the ratio of retractions to extensions, and the marked shift of cell contours over time. The destabilization of protrusions might be due to weaker integrin-mediated interaction of focal adhesions with the ECM, but also due to impaired stress-mediated maturation of focal adhesions. The fact that both under- and overactivity of the Moody pathway impair protrusion stabilization may be due to the feedback between actin-myosin and focal adhesion, which also causes the well-known biphasic response of migration speed to adhesion strength of migrating cells. While the loss of moody has no significant effects on the other parameters that were measured, the loss of loco also reduces the frequency and size of protrusions, suggesting that actin polymerization may be specifically affected by increased GPCR signaling activity. Cumulatively, these impairments in protrusion/retraction behavior lead to retarded, non-isometric growth of SPG and to the irregular cell shapes observed in moody and loco mutants (Schwabe, 2017).

Interestingly, PKA, Rho1 and MLCK have been identified as important downstream effectors of Moody signaling. All three factors are well known to control actin-myosin contraction; Rho1 and MLCK as positive regulators, PKA as a negative regulator. More recently, Rho1 activity has been shown to also drive actin polymerization at the leading edge, and a PKA-RhoGDI-Rho1 regulators feedback loop has been suggested to act as a pacemaker of protrusion- retraction cycles (Schwabe, 2017).

The role of Moody pathway signaling in directed and well-coordinated cell growth is strikingly similar to the function of trimeric G protein signaling in other contexts. In Dictyostelium, G protein signaling is essential for directed cell migration: When all G protein signaling is abolished, cells are still mobile and actively generate protrusions, however these protrusions form in random directions, with the result that the cells lose their directionality. During gastrulation in Drosophila, signaling by the G12 ortholog Concertina and the putative GPCR ligand Folded Gastrulation synchronizes apical actin-myosin constrictions of mesodermal precursor cells, and thereby effects their concerted invagination. Thus, a major role of G protein signaling during development may be to modulate basic cellular behaviors such as cell growth, protrusion, or contraction, and reduce variability within cells and between neighboring cells, with the goal of generating uniform patterns and behaviors (Schwabe, 2017).

Synchronized growth behavior of SPG is not only important for rapid epithelial closure but, ultimately, also for generating an evenly sealed BBB. All the evidence supports the notion that the defects in SJ organization that are responsible for the BBB failure are a secondary consequence of the morphogenetic function of the GPCR pathway. Cell contacts precede and are necessary for SJ formation, and the growth of cell contacts and SJ accumulation are strongly correlated. Delayed and more erratic cell- cell contact formation, as is the case in Moody pathway mutants, is likely to result in uneven circumferential distribution of SJ material later on; conversely, the timing of SJ formation per se is not affected by the pathway, arguing against a direct effect. Since the insulating function of SJs depends on their length, a decrease in the length in some local areas will result in insulation defects. Moreover, since SJs are known to form very static complexes, any irregularity in SJ distribution may be retained for long periods of time (Schwabe, 2017).

Although under- and overactivity of the Moody pathway lead to globally similar outcomes, impaired epithelium formation and failure of BBB insulation, the data point to subtly different subcellular effects of the two types of pathway modulation. During MET, loco mutants (which this study confirms as inducing pathway overactivity) show predominantly retarded growth, presumably as a result of curtailed protrusive activity, while moody mutants show severe fluctuation and variability in growth. It will be very interesting to investigate these distinct outcomes of Moody pathway misregulation in greater detail (Schwabe, 2017).

Differential expression of the Drosophila Ntan/Obek controls ploidy in the blood-brain barrier

During development tissue growth is mediated by either cell proliferation or cell growth, coupled with polyploidy. Both strategies are employed by the cell types comprising the Drosophila blood-brain barrier. During larval growth, the perineurial glia proliferate, whereas the subperineurial glia expand enormously and become polyploid. This study shows that the level of ploidy in subperineurial glia is controlled by the N-terminal asparagine amidohydrolase homolog Obek, where high Obek levels are required to limit replication. In contrast, perineurial glia express moderate levels of Obek, and increased expression blocks their proliferation. Interestingly, other dividing cells are not affected by alteration of Obek expression. In glia, Obek counteracts FGF- and Hippo-signaling to differentially affect cell growth and number. A mechanism is proposed where growth signals are integrated differentially in a glia-specific manner through different levels of Obek protein to adjust cell proliferation versus endoreplication in the blood-brain barrier (Zulbahar, 2018).

SIK suppresses neuronal hyperexcitability by regulating the glial capacity to buffer K(+) and water

Glial regulation of extracellular potassium (K(+)) helps to maintain appropriate levels of neuronal excitability. While channels and transporters mediating K(+) and water transport are known, little is understood about upstream regulatory mechanisms controlling the glial capacity to buffer K(+) and osmotically obliged water. This study identified salt-inducible kinase (SIK3) as the central node in a signal transduction pathway controlling glial K(+) and water homeostasis in Drosophila. Loss of SIK leads to dramatic extracellular fluid accumulation in nerves, neuronal hyperexcitability, and seizures. SIK3-dependent phenotypes are exacerbated by K(+) stress. SIK promotes the cytosolic localization of HDAC4, thereby relieving inhibition of Mef2-dependent transcription of K(+) and water transport molecules. This transcriptional program controls the glial capacity to regulate K(+) and water homeostasis and modulate neuronal excitability. HDAC was identified as a candidate therapeutic target in this pathway, whose inhibition can enhance the K(+) buffering capacity of glia, which may be useful in diseases of dysregulated K(+) homeostasis and hyperexcitability (Li, 2019).

Neuronal excitability is tightly coupled to complex ion dynamics in the nervous system. As ions move, so too must osmotically obliged water molecules move. Following bursts of action potentials, ionic gradients are restored via active transport. Defects in water and ion homeostasis disrupt neuronal firing and can result in edema. Potassium (K+) homeostasis is particularly important for maintaining the physiological function of neurons. Repolarization of the axonal membrane during action potentials transfers K+ ions into the extracellular space. With synchronous activity, the bulk extracellular [K+] rises, and this K+ must be rapidly buffered via glial and/or neurons lest axons depolarize, disrupting neuronal firing (Li, 2019).

Glial cells are essential for potassium and water homeostasis. Glia remove K+ ions from the extracellular space and redistribute them to areas with lower [K+] to maintain ionic gradients. Glia also express aquaporin water channels to functionally couple K+ clearance and water transport, thereby relieving osmotic stress. A number of K+ and water transport molecules mediate glial regulation of extracellular [K+], including K+ channels, Na+-K+-Cl−cotransporter 1, Na+-K+-ATPase, and aquaporin. Defects in glial transporters can result in build-up of ions in the extracellular space between axons and glia, leading to extracellular fluid accumulation. Indeed, mouse and Drosophila melanogaster mutants with defective K+transport exhibit dramatic and strikingly similar edema phenotypes in their peripheral nerves. These swellings correlate with seizure sensitivity and offer an elegant readout for identifying molecules that are required to maintain ionic and water homeostasis and a healthy level of neuronal excitability (Li, 2019).

While much is known about the key transporters and channels that mediate the flux of water and ions, the mechanisms by which glial cells regulate expression of the relevant transporters are not well understood. Delineating such regulatory mechanisms could identify approaches to leverage glial K+ and water buffering as a therapeutic strategy for neuroprotection against K+ stress-related damage. A glial-specific screen was conducted for this study in Drosophila, and a signal transduction pathway required for glial regulation of water and ion homeostasis was identified. This screen uncovered a central role for salt-inducible kinase (SIK3), a highly conserved AMP-activated protein kinase (AMPK)-family kinase that links signal sensing to changes in cellular response. Loss of SIK3 in glia results in nerve edema, neuronal hyperexcitability, and increased seizure susceptibility, all phenotypes that are commonly associated with human genetic disorders disrupting glial water and ion homeostasis. This swelling phenotype is critically and selectively sensitive to K+ stress. Moreover, this study demonstrates that SIK functions via regulation of a downstream HDAC4/Mef2 transcriptional program that controls expression of relevant ion and water transporters. HDAC is a critical negative regulator in the pathway, and pharmacological inhibition of HDAC potently suppresses the edema, hyperexcitability, and seizure phenotypes of SIK mutants. Hence, this study identifies a druggable pathway controlling the glial capacity to buffer K+ and water and a candidate therapeutic approach to achieve the long-standing goal of targeting glia for the control of hyperexcitability (Li, 2019).

Glia remove K+ from the extracellular space to maintain ionic and water homeostasis in the nervous system. Disrupting K+ buffering leads to nerve edema, neuronal hyperexcitability, and increased seizure susceptibility. In Drosophila, loss of glial K+ transport function results in characteristic focal peripheral nerve swellings due to accumulation of extracellular fluid. To identify signaling pathways required for glial regulation of K+ and water homeostasis, an in vivo glial-specific screen was performed in Drosophila. The pan-glial driver Repo-GAL was used to express a library of ~RNAi lines targeting signaling molecules, and were genes screened for that are required to prevent swelling of larval peripheral nerves. From the screen a single hit was found: glial knockdown of the gene for SIK3 results in pronounced nerve swellings (Li, 2019).

Accumulation of laminin monomers in Drosophila glia leads to glial endoplasmic reticulum stress and disrupted larval locomotion

The nervous system is surrounded by an extracellular matrix composed of large glycoproteins, including perlecan, collagens, and laminins. Glial cells in many organisms secrete laminin, a large heterotrimeric protein consisting of an α, β, and γ subunit. Prior studies have found that loss of laminin subunits from vertebrate Schwann cells causes loss of myelination and neuropathies, results attributed to loss of laminin-receptor signaling. This study demonstrates that loss of the laminin γ subunit (LanB2) in the peripheral glia of Drosophila melanogaster results in the disruption of glial morphology due to disruption of laminin secretion. Specifically, knockdown of LanB2 in peripheral glia results in accumulation of the β subunit (LanB1), leading to distended endoplasmic reticulum (ER), ER stress, and glial swelling. The physiological consequences of disruption of laminin secretion in glia included decreased larval locomotion and ultimately lethality. Loss of the γ subunit from wrapping glia resulted in a disruption in the glial ensheathment of axons but surprisingly did not affect animal locomotion. It was found that Tango1, a protein thought to exclusively mediate collagen secretion, is also important for laminin secretion in glia via a collagen-independent mechanism. However loss of secretion of the laminin trimer does not disrupt animal locomotion. Rather, it is the loss of one subunit that leads to deleterious consequences through the accumulation of the remaining subunits (Petley-Ragan, 2016).

The role of laminin and the ECM in glial wrapping has been investigated in vertebrate systems by examining the gross morphological and molecular changes induced by knocking out individual laminin subunits in glia. A decrease in glial wrapping, proliferation, and survival was attributed to a lack of laminin binding to various receptors, including integrins, dystroglycan, and syndecans. However, it was found that knock-out of laminin resulted in more severe phenotypes than the additive phenotypes of multiple-receptor knock-out studies. This paper presents evidence that eliminating one laminin subunit causes cellular changes that go beyond the loss of receptor-ligand signaling and contribute to glial mutant phenotypes (Petley-Ragan, 2016).

This study has demonstrated that glial cells expressing RNAi against three different laminin subunits result in morphological defects. Knockdown of the laminin γ subunit LanB2 results in extensively swollen glia, accumulation of LanB1 in the ER, and expanded ER, leading to ER stress with disrupted intracellular morphology, including vacuole-like structures. In laminin assembly, the β-γ dimer is formed followed by α chain recruitment where trimer formation is necessary for transport through the Golgi and secretion. Studies in vitro and in C. elegans found intracellular accumulation of the γ laminin subunit in the absence of the β subunit. However, the subcellular localization and physiological consequences of the intracellular laminin accumulations was not determined. Analysis of the knockdown of the γ subunit (LanB2) in all glia extended this line of study and found that loss of LanB2 led to β subunit (LanB1) accumulations, ER expansion, and ER stress. Glial swelling due to LanB2 knockdown was correlated with excess LanB1, and the swollen glial phenotype was partially rescued by introducing a heterozygous LanB1 deficiency in the background, effectively halving the amount of LanB1 available for translation. The partial rescue in glial swelling due to LanB1 heterodeficiency supports the hypothesis that unbound LanB1 is acting as a misfolded protein in the ER to induce the unfolded protein response and ER stress. In further support, overexpression of LanB1 alone was found to result in ER aggregates and vacuoles in glia. Knockdown of Tango1, a protein known to mediate ER exit of collagen, resulted in LanB1 and LanB2 accumulations in the ER. Tango1 knockdown results in laminin retention in Drosophila ovary follicle cells. However, the subcellular localization of laminin was not demonstrated. The current results refine these observations as both LanB2 and Tango1 knockdown result in LanB1 accumulations specifically in the expanded ER. However, while LanB2-RNAi led to severe glial swelling and locomotion defects, expression of Tango1 RNAi did not. In the case of Tango1 RNAi, this is possibly due to Tango1 knockdown inhibiting secretion of assembled laminin heterotrimers. Previously, Tango1 has been implicated primarily in collagen secretion, and this study found Tango1 mediates laminin secretion in the absence of collagen IV in perineurial glia. The findings imply that there may be a Tango1-specific binding site on laminin or that Tango1 may play a broader role in protein secretion than previously thought. Together, these findings suggest that the glial defects observed after LanB2 knockdown are due to excess amounts of unbound LanB1 monomers in the ER causing ER stress. Alternatively, the increased LanB1 and ribosomal content in these areas may indicate increased translation due to glia sensing inadequate amounts of external laminin (Petley-Ragan, 2016).

The glial defects observed are likely due to disruption of laminin secretion, but it is possible that the defects observed were in part due to loss of laminin from the extracellular space and loss of ECM receptor signaling, such as integrin signaling. The first explanation is favored as blocking the secretion of the laminin trimer by Tango1 RNAi did not result in glial swelling, suggesting that the reduction in laminin secretion did not lead to glia disruption. Loss of integrins and metalloproteinase-mediated degradation of the surrounding ECM does not lead to glial swelling or formation of vacuoles, but rather triggers a loss of perineurial glia wrapping of the axon. In Drosophila, the fat body and hemocytes contribute to the deposition of ECM proteins in the basement membrane surrounding the nervous system, explaining why the loss of laminin secretion from glia in Tango1 does not lead to the same phenotypes as integrin knockdown (Petley-Ragan, 2016).

This raises the interesting question of why Drosophila glia secrete laminin and not other ECM proteins. Similarly, why do Drosophila wrapping glia, which do not contact a basement membrane, express laminin? Wrapping glia were morphologically affected by LanB2 knockdown. However. accumulations of LanB1 within wrapping glia were not seen, implying that wrapping glia express low levels of laminin. With the available antibodies, it is difficult to detect and assess the levels of laminin between axons and wrapping glia or between wrapping and subperineurial glia. The presence of laminin interior to the nerve is supported by previous experiments where integrin knockdown in wrapping glia resulted in a lack of process extension similar to that seen after LanB2 knockdown, suggesting that integrin may bind to laminin present between axons and wrapping glia or between wrapping and subperineurial glia. Although LanB2 knockdown in wrapping glia resulted in morphological defects, including decreased wrapping, third-instar larvae exhibited no mobility defects and even survived to adulthood and this is also true of integrin knockdown in the wrapping glia (data not shown). The surprising finding that full contact between wrapping glia and peripheral axons is unnecessary for locomotion is consistent with mutants in the Na-K-Cl cotransporter, Ncc69. Ncc69 mutant larvae have significant osmotic swelling that inhibits direct contact between axons and the wrapping glia and yet these larvae had normal action potential activity and survived to adult stages. Apoptosis-induced death of the wrapping glia did, however, result in a total lack of mobility and lethality, indicating that either the wrapping glia or other nrv2-GAL4-expressing cells are essential to survival and locomotion (Petley-Ragan, 2016).

Larval survival and mobility were affected by knockdown of LanB2 in perineurial glia, a glial subtype that does not directly contact axons. Perineurial glia are born late in embryogenesis, surround the CNS and PNS during larval stages, and have been implicated in ECM remodeling during nervous system growth. This study demonstrates that perineurial glia secrete laminin, but not collagen IV, and contribute to nervous system function. The decreased mobility seen in 46F>LanB2-RNAi larvae provides support for the role of the outer layer of perineurial glia and the ECM in structurally supporting the larval nervous system as these glia do not contact either neuronal cell bodies or axons (Petley-Ragan, 2016).

Ultimately, the current findings suggest that loss of receptor-ligand signaling is not the sole cause of glial morphological and physiological defects due to loss of laminins. Due to the conserved structure and function of glia across the animal kingdom, it is likely that Schwann cells lacking one subunit of laminin or with a mutation in one subunit affecting laminin trimerization could also demonstrate morphological and functional defects due to the accumulation of unbound laminin subunits leading to ER stress. This could have significant implications as ER stress is important in the pathogenesis of various diseases affecting myelination in the CNS and PNS. This hypothesis has not yet been investigated in vertebrate glia, but it may provide insight into the reasons decreased glial wrapping, proliferation, and survival are seen in vertebrates after knock-out or with inherited mutations in laminin subunits. Schwann cell-specific deletion of Lamc1 (the γ-1 subunit) leads to a loss of α and β subunit expression and disruption of basement membrane secretion. Loss of γ-1 leads to defects in radial sorting and myelination by myelinating Schwann cells, as well as to loss of proliferation and blocked differentiation in the premyelination stage. Similarly loss of γ-1 leads to the absence of nonmyelinating Schwann cells and the loss of Remak bundles. The wrapping glia of Drosophila are most similar to nonmyelinating Schwann cells and in these cells loss of the γ subunit led to wrapping defects and ER stress. Whether the loss of the vertebrate γ-1 subunit leads to ER stress that results in the loss of nonmyelinating Schwann cells has not been determined. However, the current findings may represent a broader trend in the cellular response to unequal expression of related subunits that together comprise a multimeric protein, such as laminin. Future studies should focus on the effects of ER stress due to mutations in individual subunits of laminin in Schwann cells and other tissues (Petley-Ragan, 2016).

A miRNA screen procedure identifies garz as an essential factor in adult glia functions and validates Drosophila as a beneficial 3Rs model to study glial functions and GBF1 biology

To study the influence of adult glial cells in ageing flies, a genetic screen was performed in Drosophila using a collection of transgenic lines providing conditional expression of micro-RNAs (miRNAs). This study describes a methodological algorithm to identify and rank genes that are candidate to be targeted by miRNAs that shorten lifespan when expressed in adult glia. Four different databases were used for miRNA target prediction in Drosophila but little agreement was found between them, overall. However, top candidate gene analysis shows potential to identify essential genes involved in adult glial functions. One example from the top candidates' analysis is gartenzwerg (garz). It was established that garz is necessary in many glial cell types, that it affects motor behaviour and, at the sub-cellular level, is responsible for defects in cellular membranes, autophagy and mitochondria quality control. The remarkable conservation of functions between garz and its mammalian orthologue, GBF1, was verified validating the use of Drosophila as an alternative 3Rs-beneficial model to knock-out mice for studying the biology of GBF1, potentially involved in human neurodegenerative diseases (Goncalves-Pimentel, 2020)

Fibroblast growth factor signaling instructs ensheathing glia wrapping of Drosophila olfactory glomeruli

The formation of complex but highly organized neural circuits requires interactions between neurons and glia. During the assembly of the Drosophila olfactory circuit, 50 olfactory receptor neuron (ORN) classes and 50 projection neuron (PN) classes form synaptic connections in 50 glomerular compartments in the antennal lobe, each of which represents a discrete olfactory information-processing channel. Each compartment is separated from the adjacent compartments by membranous processes from ensheathing glia. This study shows that Thisbe, an FGF released from olfactory neurons, particularly from local interneurons, instructs ensheathing glia to wrap each glomerulus. The Heartless FGF receptor acts cell-autonomously in ensheathing glia to regulate process extension so as to insulate each neuropil compartment. Overexpressing Thisbe in ORNs or PNs causes overwrapping of the glomeruli their axons or dendrites target. Failure to establish the FGF-dependent glia structure disrupts precise ORN axon targeting and discrete glomerular formation (Wu, 2017)

The use of discrete neuropil compartments for organizing and signaling information is widespread in invertebrate and vertebrate nervous systems. In both the fly antennal lobe and vertebrate olfactory bulb, axons from different ORN classes are segregated into distinct glomeruli. The rodent barrel cortex also uses discrete compartments, the barrels, to represent individual whiskers. This study shows that FGF signaling between neurons and glia mediates neural compartment formation in the Drosophila antennal lobe (Wu, 2017)

Members of the FGF family have diverse functions in a variety of tissues in both vertebrates and invertebrates. Vertebrate FGFs regulate not only neural proliferation, differentiation, axon guidance, and synaptogenesis but also gliogenesis, glial migration, and morphogenesis. Many of these roles are conserved in invertebrates. For example, Ths and Pyr induce glial wrapping of axonal tracts, much like the role other FGF members play in regulating myelin sheaths in mammals. Ths and Pyr also control Drosophila astrocyte migration and morphogenesis; likewise, FGF signaling promotes the morphogenesis of mammalian astrocytes. Therefore, studying the signaling pathways in Drosophila will extend understanding of the principles of neural development (Wu, 2017)

In ensheathing glia, whose developmental time course and mechanisms have not been well documented before this study, a glial response was observed to FGF signaling reminiscent of the paradigm shown previously; however, the exquisite compartmental structure of the Drosophila antennal lobe and genetic access allowed this study to scrutinize further the changes of neuropil structure and projection patterns that occurred alongside morphological phenotypes in ensheathing glia. The requirement for Ths in LNs was demonstrated, although it is possible that ORNs and PNs also contribute. The function was tested of the other ligand, Pyr, in antennal lobe development. No change was detected in ensheathing glia morphology with pyr RNAi, and double RNAi against ths and pyr did not enhance the phenotype compared with ths knockdown alone (Wu, 2017)

FGF signaling in glomerular wrapping appears to be highly local. In overexpression experiments, the hyperwrapping effect was restricted to the glomerulus where the ligand is excessively produced and did not spread to nearby nonadjacent glomeruli. These experiments suggest that Ths communicates locally to instruct glial ensheathment of the glomeruli rather than diffusing across several microns to affect nearby glomeruli. Because heparan sulfate proteoglycans are known to act as FGF coreceptors by modulating the activity and spatial distribution of the ligands, it is speculated that Ths in the antennal lobe may be subject to such regulation to limit its diffusion and long-range effect (Wu, 2017)

The data showed that deficient ensheathment of antennal lobe glomeruli is accompanied by imprecise ORN axon targeting. However, it was not possible to determine whether these targeting defects reflect initial axon-targeting errors or a failure to stabilize or maintain the discrete targeting pattern. Previous models for the establishment of antennal lobe wiring specificity suggested that the glomerular map is discernable by the time glia processes start to infiltrate the antennal lobe. Because of a lack of class-specific ORN markers for early developmental stages, the relative timing between when neighboring ORN classes refine their axonal targeting to discrete compartments and when ensheathing glia barriers are set up still remains unclear. Nevertheless, this discovery that FGF signaling functions in the formation of discrete neuronal compartments in the antennal lobe highlights an essential role for glia in the precise assembly of neural circuits (Wu, 2017)

Wrapping glial morphogenesis and signaling control the timing and pattern of neuronal differentiation in the Drosophila lamina

Various regions of the developing brain coordinate their construction so that the correct types and numbers of cells are generated to build a functional network. Previous work has discovered that wrapping glia in the Drosophila visual system are essential for coordinating retinal and lamina development. Wrapping glia, which ensheath photoreceptor axons, respond to an epidermal growth factor cue from photoreceptors by secreting insulins. Wrapping glial insulins activate the mitogen-activated protein kinase (MAPK) pathway downstream of insulin receptor in lamina precursors to induce neuronal differentiation. The signaling relay via wrapping glia introduces a delay that allows the lamina to assemble the correct stoichiometry and physical alignment of precursors before differentiating and imposes a stereotyped spatiotemporal pattern that is relevant for specifying the individual lamina neuron fates. This study further describes how wrapping glia morphogenesis correlates with the timing of lamina neuron differentiation by 2-photon live imaging. Although MAPK activity in lamina precursors drives neuronal differentiation, the upstream receptor driving MAPK activation in lamina precursors and the ligand secreted by wrapping glia to trigger it differentially affect lamina neuron differentiation. These results highlight differences in MAPK signaling properties and confirm that communication between photoreceptors, wrapping glia, and lamina precursors must be precisely controlled to build a complex neural network (Rossi, 2018).

Cortex glia clear dead young neurons via Drpr/dCed-6/Shark and Crk/Mbc/dCed-12 signaling pathways in the developing Drosophila optic lobe

The molecular and cellular mechanism for clearance of dead neurons was explored in the developing Drosophila optic lobe. During development of the optic lobe, many neural cells die through apoptosis, and corpses are immediately removed in the early pupal stage. Most of the cells that die in the optic lobe are young neurons that have not extended neurites. This study shows that clearance was carried out by cortex glia via a phagocytosis receptor, Draper (Drpr). drpr expression in cortex glia from the second instar larval to early pupal stages was required and sufficient for clearance. Drpr that was expressed in other subtypes of glia did not mediate clearance. Shark and Ced-6 mediated clearance of Drpr. The Crk/Mbc/dCed-12 pathway was partially involved in clearance, but the role was minor. Suppression of the function of Pretaporter, CaBP1 and phosphatidylserine delayed clearance, suggesting a possibility for these molecules to function as Drpr ligands in the developing optic lobe (Nakano, 2019).

Many studies have explored the cellular and molecular mechanisms for clearance of dead neurons in the developing Drosophila CNS. During embryonic development, dead neurons are phagocytosed by subperineurial glia. Draper (Drpr) acts as a phagocytosis receptor on the glial membrane to clear dead neurons in the embryo. Another receptor, Six-microns-under (SIMU), works in cortex glia to allow recognition and engulfment of apoptotic cells, whereas Drpr works to degrade apoptotic cells in the embryonic CNS. During metamorphosis, dead neurons are engulfed by glia in the CNS. Elimination of neurites of vCrz neurons during metamorphosis is performed by astrocyte-like glia via the Crk/Mbc/dCed-12 signaling pathway but not the Drpr pathway. In contrast, elimination of cell bodies of vCrz neurons, a group of neurons that express neuropeptide Corazonin, requires Drpr, but its expression is not required in astrocyte-like glia. However, recent studies have reported inconsistent results on the requirement of Drpr for dead cell clearance and the glia subtypes that work for clearance in the brain during metamorphosis. It has been reported that dead neurons that died in the central brain before the beginning of the third larval instar and in the optic lobe before the late third larval instar are cleared by cortex glia via the Drpr pathway, but neurons that die thereafter are efficiently cleared without Drpr. Drpr has been shown to be required for apoptotic cell clearance during metamorphosis and its expression is required in ensheathing glia and astrocyte-like glia, but not in cortex glia (Nakano, 2019).

One of the causes of inconsistency among previous studies may be differences in the cellular materials to be phagocytosed, and different mechanisms could work for phagocytosis of different materials in the CNS during metamorphosis. Three types of neurons need to be phagocytosed during metamorphosis. Obsolete larval neurons die, and their cell bodies and neurites are removed by phagocytosis. Larval neurons of another type are respecified from larval to adult neurons via pruning of larval neurites and extension of new adult neurites. Pruned neurites are removed by phagocytosis. Adult-specific neurons are produced by precursor cells during post-embryonic development and differentiate during metamorphosis. A number of these young neurons die during development before extending neurites. Therefore, studies on a single type of neuron or specifically defined neurons are needed to define the molecular and cellular mechanisms for clearance of dead neurons. Moreover, clearance of neurites and cell bodies of dead neurons should be studied independently (Nakano, 2019).

In this study, clearance of dead neurons in the developing optic lobe was examined. The Drosophila optic lobe is a unique center in which a large number of dying cells are observed during its development. Most dying neurons in the optic lobe are young neurons that had just started to differentiate into adult neurons. One of paired neurons derived from intermediate precursors (GMCs) is eliminated by apoptosis under the control of Notch signaling. Neurons that die in the developing optic lobe have not yet extended neurites at the time they die. Therefore, cellular materials to be cleared after the cell death include nuclei and general cytoplasm, but not neurites in the developing optic lobe (Nakano, 2019).

The adult optic lobe develops from the primordium during metamorphosis. Optic lobe neurons are produced by two proliferation centers, the outer proliferation center (OPC) and inner proliferation center (IPC). Neurons differentiate, extend neurites, and produce four types of neuropil, the lamina, medulla, lobula plate, and lobula. Then, the optic lobe consists of four types of neuropil and surrounding cortices of neuronal cell bodies. According to previous studies, many neurons and a small number of precursor cells undergo cell death during optic lobe development. This cell death does not occur randomly in the optic lobe but occurs in clusters in a specific temporal and spatial pattern. The number of dead cells in the optic lobe starts to increase at the puparium formation, reaches a peak at 24 h after puparium formation (24 h APF), and decreases to almost zero by 48 h APF. Two types of cell death are involved in this process: ecdysone dependent and independent. Both types of cell death are apoptosis and involve the Drosophila effector caspases, DrIce and Dcp-1. DrIce plays an important role in dead cell clearance as well. The role of cell death is to prevent the emergence of abnormal neural structures in the optic lobe (Nakano, 2019).

This study explored the cellular and molecular mechanisms for clearance of dead young neurons in the developing optic lobe. The results showed that clearance was carried out by cortex glia via a phagocytosis receptor, Drpr. Drpr expression in cortex glia from the second instar larval to early pupal stages was required and sufficient for clearance. Signaling molecules, Shark and Ced-6 mediated clearance downstream of Drpr. The Crk/Mbc/dCed-12 pathway was partially involved in clearance, but the role was minor. Suppression of the function of Pretaporter, CaBP1 and phosphatidylserine delayed clearance, suggesting a possibility for these molecules to function as Drpr ligands in the developing optic lobe (Nakano, 2019).

This study revealed that Drpr expressed in cortex glia were required for dead cell clearance in the MLpL region of the developing optic lobe, and that Drpr in other subtypes of glia did not mediate clearance. This is the first study that showed clearance of dead young neurons in the developing optic lobe required Drpr expression in the cortex glia. In the lamina region, lamina distal cortex glia work for dead cell clearance (Nakano, 2019).

The expression pattern of Drpr agreed with the alteration in the activity of dead cell clearance in the optic lobe during metamorphosis. At early pupal stages, Drpr is expressed weakly in a mesh-like pattern and strongly in a centripetal pattern in cortex glia in the MLpL region. This expression weakened thereafter, and only weak expression was seen in a mesh-like pattern during the last half of the pupal period. Moreover, no protrusion was seen of Drpr expressing glial cytoplasm into the neuropil from neuropil glia (NG) at early pupal stages. This agrees with the fact that cell death in the developing optic lobe occurs mainly in young neurons before they extend neurites or in abnormal neurons with no neurites (Nakano, 2019).

After 48 h APF, cell death was rarely observed and thus activity of dead cell clearance was low. However, this does not mean that glia lost potential ability to clear corpses at late pupal stages. Forced expression of wild-type drpr on the drpr mutant background at 48 or 72 h APF resulted in clearance of accumulated TUNEL-positive cells. This indicates that the components of the mechanism for dead cell clearance except Drpr are retained until late pupal stages. Therefore, if some cells died at late pupal stages and Drpr expression was induced in cortex glia, the dead cells would be cleared via Drpr pathway. Moreover, the fact that accumulated TUNEL-positive cells were removed when wild-type drpr was forcibly expressed in late pupal stages on the drpr mutant background suggests that 'eat me' signals were secreted or displayed by accumulated TUNEL-positive cells in drpr mutants not only at early pupal stages, when the cells died, but also at late pupal stages long after cell death (Nakano, 2019).

At late pupal stages, strong Drpr expression appeared in the cytoplasmic protrusions from the neuropil glia (NG), and astrocyte-like glia simultaneously started expressing molecular markers (specific GAL4s). This Drpr was not utilized for clearance of dead neurons, as almost no cell death arises at this stage in control conditions. In the neuropil of the optic lobe at late pupal stages, neurites extend and form synapses to make and complete neural networks. When new synapses are formed during development of the Drosophila larval neuromuscular junction, significant amounts of presynaptic membranes and a subset of immature synapses are removed from the junction by surrounding glia and postsynaptic muscle via the Drpr/dCed-6 pathway (Fuentes-Medel, 2009). Thus, the same process may arise at developing synapses in the developing optic lobe, and astrocyte-like glia expressing Drpr in the cytoplasmic protrusions may function to remove unnecessary presynaptic membranes and immature synapses (Nakano, 2019).

Previous studies have reported that astrocyte-like glia are responsible for clearance of degenerating axons of dying obsolete larval neurons in the ventral nerve cord (Tasdemir-Yilmaz, 2014) and of pruned axons of γ neurons in the mushroom body. In contrast, degenerating axons are removed by ensheathing glia in the olfactory lobe following Wallerian degeneration of the olfactory nerve. Therefore, different subtypes of glia work to clear degenerating axons in different contexts. Astrocyte-like glia may specifically function for clearance of 'programmed' degenerating axons and ensheathing glia for clearance of 'accidently' degenerating axons. In addition, another subtype of glia, cortex glia, functions to remove dead young neurons. These young neurons had just started to differentiate into adult neurons in the developing optic lobe and have not yet extended neurites at the time they die. Tasdemir-Yilmaz (2014) reported that elimination of cell bodies of obsolete vCrz neurons requires Drpr, but its expression is not required in astrocyte-like glia. Young neurons in the optic lobe and cell bodies of obsolete vCrz neurons in the ventral nerve cord both locate in the cortex and almost the same cellular materials are cleared after the cell death, including nucleus and general cytoplasm, but not neurites. Therefore, as with dead young neurons in the optic lobe, the expression of Drpr in cortex glia would be required for clearance of cell bodies of dead vCrz neurons. Comparative studies are expected in the future on the mechanisms for clearance of degenerating axons of dead neurons, degenerating axons of cut nerves, dead young neurons, and cell bodies of dead obsolete neurons. Moreover, considering that 'accidently' degenerating axons are cleared by a different subtype of glia from 'programmed' degenerating axons, a possibility should be tested that cell bodies of neurons that died 'accidently' are cleared by a distinct subtype of glia (Nakano, 2019).

This is the first study to reveal that Shark mediates Drpr-dependent clearance of dead neurons in the CNS. Moreover, this study suggests that Ced-6, Crk/Mbc/dCed-12, and Rac1 are partially involved in clearance of dead young neurons. Therefore, both Drpr/Shark/dCed-6 and Crk/Mbc/dCed-12 pathways work for dead cell clearance in the developing optic lobe. As cortex glia function for clearance of dead neurons in the developing optic lobe, these pathways must work in cortex glia (Nakano, 2019).

A previous study reported that these pathways function to mediate removal of degenerating axons in ensheathing glia in the adult olfactory lobe when the olfactory nerve is cut (Ziegenfuss, 2012). The same pathways work in astrocyte-like glia around the mushroom body when axons of γ neurons are pruned, and in the ventral nerve cord when neurites of dead vCrz neurons are cleared during metamorphosis (Tasdemir-Yilmaz, 2014). Therefore, Drpr/Shark/dCed-6 and Crk/Mbc/dCed-12 pathways generally function to clear corpses in different glia in different contexts. However, the relative role played by each pathway depends on the situation. Although drpr mutation strikingly inhibited dead cell clearance in the optic lobe, knockdown of the Crk/Mbc/dCed-12 pathway had only a moderate effect. In contrast, removal of olfactory nerve axons that have undergone Wallerian degeneration is strongly affected by both drpr mutation and Crk/Mbc/dCed-12 knockdown (Ziegenfuss, 2012). In the pruning of mushroom body γ axons, mutation of drpr and knockdown of Crk/Mbc/dCed-12 additively affect clearance, although mutation of drpr has a stronger effect (Tasdemir-Yilmaz, 2014). In the removal of axons from dead vCrz neurons during metamorphosis, knockdown of dCed-12 causes a moderate defect, whereas drpr mutation causes no defect by itself but only enhances the defect caused by dCed-12 knockdown (Tasdemir-Yilmaz\, 2014). How the relative role of these pathways is regulated and why remain to be defined (Nakano, 2019).

Previous studies reported that Pretaporter, CaBP1 and phosphatidylserine act as Drpr ligands when dead embryonic cells are phagocytosed in the Drosophila embryo and cultured cells. The present study suggested a possibility that these molecules mediate signaling for dead cell clearance as a Drpr ligand in the developing optic lobe. However, Pretaporter and CaBP1 are not essential for clearance and their role would be minor. Therefore, relative role of molecules that work for dead cell clearance as ligands for Drpr may be different depending on the context. As described above, different subtypes of glia work to clear corpse in the CNS: ensheathing glia for Wallerian's degenerating axons, astrocyto-like glia for pruned axons and degenerating axons of dead vCrz neurons, and cortex glia for dead young neurons in the optic lobe. Therefore, it is to be defined whether difference in ligand molecules is involved in activating different subtypes of glia (Nakano, 2019).

The present study agrees with results described by Tasdemir-Yilmaz (2014), who reported that Drpr is required for elimination of cell bodies of vCrz neurons that die at 3-7 h APF. However, the current study disagrees with other studies (Nakano, 2019 and references therein).

Several possible causes may have led to these inconsistencies. One possibility is the difference in cellular materials to be cleared, that is, cell bodies or neurites, as mentioned earlier. The present study examined dead young neurons in the developing optic lobes. Cellular materials to be cleared include only nucleus and general cytoplasm, but not neurites. Tasdemir-Yilmaz (2014) studied vCrz neurons and found that molecular mechanisms for clearance of cell bodies and neurites are different. However, other studies examined the central brain or the whole brain, which include cell bodies of dead neurons, neurites of dead neurons and pruned neurites. Another possibility is the difference in methods to detect dead neurons. This study used the ABC TUNEL method, which detects degraded DNA in dead cell nuclei with the streptavidin-biotin-peroxidase complex (Vector Laboratories). This method is far more sensitive and reliable than the fluorescent TUNEL method (compare the number of TUNEL-positive cells between the present study and other studies). Another method to detect dead cells is anti-Dcp-1 antibody staining. This method has some problems with detection of accumulated dead cell corpses in phagocytosis-defective mutants. It detects activated Dcp-1, one of the effector caspases. However, another effector caspase, DrIce, is also expressed and is a more effective inducer of apoptosis than Dcp-1. Therefore, this method may not detect all dead cells. Another problem with this method is the unknown stability of activated Dcp-1 in dead cells. Therefore, detection of activated Dcp-1 does not show exactly how many dead cells have accumulated in phagocytosis-defective mutants. Moreover, when dendrites are pruned during remodeling of dendritic arborization sensory neurons during metamorphosis, caspase activity is detected in the dendrite. This suggests that the anti-Dcp-1 antibody may detect pruned dendrites as well as dead cells. Finally, the subtypes of glia that clear corpses are also different in the present study and previous ones. This study found that expression of GAL4 in glia subtype-specific GAL4 lines drastically changed during metamorphosis, and the expression pattern at pupal stages was different from adult stages in many GAL4 lines. However, previous studies did not examine the expression pattern of the GAL4 lines they used. Altogether, studies on a single type of dead neuron or identified neurons are required in the future. The mechanisms for clearance of dead cell bodies and degenerating neurites should be studied independently. The expression pattern of GAL4 lines in subtypes of glia should be carefully assessed before using the line as a GAL4 driver (Nakano, 2019).

The secreted neurotrophin Spatzle 3 promotes glial morphogenesis and supports neuronal survival and function

Most glial functions depend on establishing intimate morphological relationships with neurons. Significant progress has been made in understanding neuron-glia signaling at synaptic and axonal contacts, but how glia support neuronal cell bodies is unclear. This study explored the growth and functions of Drosophila cortex glia (which associate almost exclusively with neuronal cell bodies) to understand glia-soma interactions. Cortex glia were shown to tile with one another and with astrocytes to establish unique central nervous system (CNS) spatial domains that actively restrict glial growth, and selective ablation of cortex glia causes animal lethality. In an RNAi-based screen, alphaSNAP (soluble NSF [N-ethylmalemeide-sensitive factor] attachment protein alpha) and several components of vesicle fusion and recycling machinery were identified as essential for the maintenance of cortex glial morphology and continued contact with neurons. Interestingly, loss of the secreted neurotrophin Spatzle 3 (Spz3) phenocopied alphaSNAP phenotypes, which included loss of glial ensheathment of neuron cell bodies, increased neuronal cell death, and defects in animal behavior. Rescue experiments suggest that Spz3 can exert these effects only over very short distances. This work identifies essential roles for glial ensheathment of neuronal cell bodies in CNS homeostasis as well as Spz3 as a novel signaling factor required for maintenance of cortex glial morphology and neuron-glia contact (Coutinho-Budd, 2017).

Tyramine actions on Drosophila flight behavior are affected by a glial dehydrogenase/reductase

The biogenic amines octopamine (OA) and tyramine (TA) modulate insect motor behavior in an antagonistic manner. OA generally enhances locomotor behaviors such as Drosophila larval crawling and flight, whereas TA decreases locomotor activity. However, the mechanisms and cellular targets of TA modulation of locomotor activity are incompletely understood. This study combines immunocytochemistry, genetics and flight behavioral assays in the Drosophila model system to test the role of a candidate enzyme for TA catabolism, named Nazgul (Naz), in flight motor behavioral control. It is hypothesized that the dehydrogenase/reductase Naz represents a critical step in TA catabolism. Immunocytochemistry reveals that Naz is localized to a subset of Repo positive glial cells with cell bodies along the motor neuropil borders and numerous positive Naz arborizations extending into the synaptic flight motor neuropil. RNAi knock down of Naz in Repo positive glial cells reduces Naz protein level below detection level by Western blotting. The resulting consequence is a reduction in flight durations, thus mimicking known motor behavioral phenotypes as resulting from increased TA levels. In accord with the interpretation that reduced TA degradation by Naz results in increased TA levels in the flight motor neuropil, the motor behavioral phenotype can be rescued by blocking TA receptors. These findings indicate that TA modulates flight motor behavior by acting on central circuitry and that TA is normally taken up from the central motor neuropil by Repo-positive glial cells, desaminated and further degraded by Naz (Ryglewski, 2017).

The glia-geuron lactate shuttle and elevated ROS promote lipid synthesis in neurons and lipid droplet accumulation in glia via APOE/D

Elevated reactive oxygen species (ROS) induce the formation of lipids in neurons that are transferred to glia, where they form lipid droplets (LDs). This study shows that glial and neuronal monocarboxylate transporters (MCTs), fatty acid transport proteins (FATPs), and apolipoproteins are critical for glial LD formation. MCTs enable glia to secrete and neurons to absorb lactate, which is converted to pyruvate and acetyl-CoA in neurons. Lactate metabolites provide a substrate for synthesis of fatty acids, which are processed and transferred to glia by FATP and apolipoproteins. In the presence of high ROS, inhibiting lactate transfer or lowering FATP or apolipoprotein levels decreases glial LD accumulation in flies and in primary mouse glial-neuronal cultures. Human APOE can substitute for a fly glial apolipoprotein, and APOE4, an Alzheimer's disease susceptibility allele, is impaired in lipid transport and promotes neurodegeneration, providing insights into disease mechanisms (Liu, 2017).

Neurodegeneration is often characterized by cellular hallmarks such as mitochondrial dysfunction, oxidative stress, protein aggregates, proteasome or autophagosome dysfunction, and endolysosomal defects. Previous work has shown that lipid droplets (LDs), a neutral lipid-storing organelle, arise due to elevated levels of reactive oxygen species (ROS) and may be used as an early biomarker for the onset of neurodegeneration (Liu, 2015). These LDs accumulate because of mitochondrial defects that lead to highly elevated ROS, which induce the production of lipids in neurons and their subsequent transfer to glia. However, the mechanisms of lipid production or transport in the nervous system and the metabolic cooperation between neurons and glia, in both health and disease, remain poorly understood (Liu, 2017).

Previous work has found that elevating ROS in neurons alone is sufficient for glia to accumulate LDs, suggesting a transfer of lipids (or their precursors) from one cell to the other (Liu, 2015). The mechanism of lipid transfer in the nervous system has not been previously studied. Moreover, although the resting brain's energy expenditure is significantly higher than that of most organs, the cellular appropriation of neuronal energy and its derivation is unclear. In mammals, blood glucose is the brain's primary fuel source. However, metabolic intermediates, such as lactate and ketone bodies, can also be used as a source of energy. Along these lines, the Astrocyte Neuron Lactate Shuttle (ANLS) Hypothesis was shown to play a role of nervous system homeostasis in Drosophila (Volkenhoff, 2015) and mice. In Drosophila, perineural glia possess the enzymes necessary to metabolize the sugar trehalose, whose metabolites, including lactate, are secreted by glia and thought to be taken up by neurons (Volkenhoff, 2015). Similarly, in mammals, spectroscopy studies have shown that circulating blood glucose is taken up by astrocytes and hypothesized to be converted to lactate. Given that monocarboxylate transporters (MCTs) are expressed in the Drosophila (Volkenhoff, 2015) and mammalian nervous systems, it is thought that lactate is transported from glia to neurons through these membrane carriers. However, the in vivo function of lactate, its transport and metabolism in neurons and glia, and its relationship to LD remain to be determined (Liu, 2017).

Lipid transporters are likely to play a role in the nervous system metabolic homeostasis. These include Apolipoprotein E (ApoE) and Apolipoprotein D (ApoD). ApoE assists in circulating lipoprotein formation and is assumed to function in lipid transport in the brain. ApoE-deficient mice (Apoe-/-) exhibit early-onset hyperlipidemia and aortic plaque formation. However, there are conflicting reports regarding brain morphological changes in Apoe-/- mice with respect to synaptic loss and cytoskeletal changes. Humans have three allelic variants of ApoE, resulting in six possible allelic combinations that alter an individual's likelihood of developing hyperlipoproteinemia (APOE2) or Alzheimer's disease (AD) (APOE4). Indeed, 40%-80% of patients with AD carry at least one copy of the APOE4 allele, and homozygous carriers have a greater than 50% lifetime risk of developing AD. APOE4 is by far the most common AD susceptibility locus. The APOE3 allele is the predominant allele in the population and confers an average risk for AD, whereas APOE2 is considered 'protective,' as it decreases an individual's risk for developing AD. The importance of APOE in AD suggests that it plays a major role in facilitating proper nervous system function. However, the physiological function of ApoE in metabolism of neurons and glia is poorly characterized, and its function in relation to LD metabolism has not been documented (Liu, 2017).

Although certain lipid metabolism proteins are highly conserved in flies and mammals, including Sterol Regulatory Element Binding Protein (SREBP), ApoE does not appear to have a direct ortholog in Drosophila. In flies, the ApoD homologs glial lazarillo (Glaz) and neural lazarillo (Nlaz) are protective against stress. Loss of Glaz or Nlaz leads to elevated sensitivity to ROS and a decrease in the triglyceride content of the whole animal, suggesting that ApoD provides a protection against certain stressors (Liu, 2017).

This study has explored the sources of energy and lipids that lead to LD accumulation in glia by selectively manipulating the expression and function of genes and proteins separately in Drosophila photoreceptor neurons and glia. Evidence is provided that supporting the ANLS and demonstrate that lactate transport from glia to neuron through monocarboxylate transporters affects neuronal lipogenesis. In response to elevated levels of ROS, neuronal lipids are transferred to glia, where they are stored as LD. Lactate-derived neuronal lipid transport to glia depends on fatty acid transport proteins (FATPs) and apolipoproteins in flies and mice. This study has also documented that the human APOE2 and APOE3 alleles can functionally substitute for the loss of Glaz in flies to permit lipid transport from neurons to glia, whereas APOE4 cannot. The inability of APOE4 flies to accumulate LD in response to ROS presages neuronal degeneration and loss. Likewise, when subjected to high ROS, neuron-glia co-cultured mouse primary cells that lack APOE are unable to form LD. It is proposed that LD formation in glia requires ApoE and that LD accumulation protects against neurodegeneration by scavenging peroxidated lipids (Liu, 2017).

Chaski, a novel Drosophila lactate/pyruvate transporter required in glia cells for survival under nutritional stress

The intercellular transport of lactate is crucial for the astrocyte-to-neuron lactate shuttle (ANLS), a model of brain energetics according to which neurons are fueled by astrocytic lactate. This study shows that the Drosophila chaski gene encodes a monocarboxylate transporter protein (MCT/SLC16A) which functions as a lactate/pyruvate transporter, as demonstrated by heterologous expression in mammalian cell culture using a genetically encoded FRET nanosensor. chaski expression is prominent in the Drosophila central nervous system and it is particularly enriched in glia over neurons. chaski mutants exhibit defects in a high energy demanding process such as synaptic transmission, as well as in locomotion and survival under nutritional stress. Remarkably, locomotion and survival under nutritional stress defects are restored by chaski expression in glia cells. These findings are consistent with a major role for intercellular lactate shuttling in the brain metabolism of Drosophila (Delgado, 2018).

Differing strategies despite shared lineages of motor neurons and glia to achieve robust development of an adult neuropil in Drosophila

In vertebrates and invertebrates, neurons and glia are generated in a stereotyped manner from neural stem cells, but the purpose of invariant lineages is not understood. This study shows that two stem cells that produce leg motor neurons in Drosophila also generate neuropil glia, which wrap and send processes into the neuropil where motor neuron dendrites arborize. The development of the neuropil glia and leg motor neurons is highly coordinated. However, although motor neurons have a stereotyped birth order and transcription factor code, the number and individual morphologies of the glia born from these lineages are highly plastic, yet the final structure they contribute to is highly stereotyped. It is suggested that the shared lineages of these two cell types facilitate the assembly of complex neural circuits and that the two birth order strategies-hardwired for motor neurons and flexible for glia-are important for robust nervous system development, homeostasis, and evolution (Enriquez, 2018).

Multiple lineages enable robust development of the neuropil-glia architecture in adult Drosophila

Neural remodeling is essential for the development of a functional nervous system and has been extensively studied in the metamorphosis of Drosophila. Despite the crucial roles of glial cells in brain functions, including learning and behavior, little is known of how adult glial cells develop in the context of neural remodeling. This study shows that the architecture of neuropil-glia in the adult Drosophila brain, which is composed of astrocyte-like glia (ALG) and ensheathing glia (EG), robustly develops from two different populations in the larva: the larval EG and glial cell missing-positive (gcm+) cells. Whereas gcm+ cells proliferate and generate adult ALG and EG, larval EG dedifferentiate, proliferate and redifferentiate into the same glial subtypes. Each glial lineage occupies a certain brain area complementary to the other, and together they form the adult neuropil-glia architecture. Both lineages require the FGF receptor Heartless to proliferate, and the homeoprotein Prospero to differentiate into ALG. Lineage-specific inhibition of gliogenesis revealed that each lineage compensates for deficiency in the proliferation of the other. Together, the lineages ensure the robust development of adult neuropil-glia, thereby ensuring a functional brain (Kato, 2020).

The neuropil-glial architectures in larval ventral nerve cords (VNC) and brains are composed of a small number of neuropil-glia, generated during the embryonic stage, whereas a large number of neuropil-glia form the glial architecture in the adult VNC (Enriquez, 2018) and brain (Awasaki, 2008; Kremer, 2017). The neuropil-glial architecture appears to change in concert with the remodeling of the brain. One group proposed a model for the generation of the adult neuropil-glia architecture, in which both larval ALG and EG undergo programmed cell death. Others reported that the cell bodies of larval ALG persist during pupal life, and they re-infiltrate their fine process into the neuropil at the late pupal stage. Neuropil-glia for an adult brain are generated from a small number of specific larval neuroblasts, termed as type II neuroblasts. However, they are not accountable for the entire architecture of neuropil-glia in an adult brain; the superiormost and the inferior regions of a brain remain uncovered by neuropil-glia generated by type II neuroblasts. Thus, a broad conceptual view of how the architecture of neuropil-glia undergoes remodeling remains to be elucidated (Kato, 2020).

This study has investigated the fate of larval ALG, EG and glial cell missing-positive (gcm+) cells, and found that the larval EG dedifferentiate, proliferate and redifferentiate into adult ALG and EG. Together with the gcm+ lineage, the larval EG lineage generates the adult neuropil-glia architecture. Finally, to investigate the interaction between the lineages in the development of the neuropil-glial architecture, this study evaluated whether one lineage compensates for the failure of gliogenesis in the other lineage (Kato, 2020).

The adult architecture of neuropil-glia is formed from two lineages: the differentiated larval EG lineage and the gcm+ lineage. Both lineages require htl for proliferation and Pros for differentiation of ALG. Each lineage compensates for the failure of the other to proliferate. Thus, the architecture of adult neuropil-glia develops robustly to ensure a functional adult brain (Kato, 2020).

Previous studies have suggested that the adult neuropil-glia are derived from larval neuroblasts, and larval neuropil-glia (both L-EG and L-ALG) undergo programmed cell death during metamorphosis. Given that neuroblasts give rise to gcm+ cells, which then generate mature glial cells, the fate of gcm+ cells was traced and were shown to generate adult neuropil-glia. Although this result is mostly consistent with previous reports, the area occupied by adult neuropil-glia derived from gcm+ cells was larger than that occupied by type II neuroblast-derived glia, as reported by Ren (2018). The results suggest that type II neuroblasts are not the sole origin of gcm+ cells that generate adult neuropil-glia. Furthermore, this study demonstrates that L-EG also participates in the genesis of adult neuropil-glia. Collectively, this study demonstrates that adult neuropil-glia are generated from gcm+ cells and L-EG (Kato, 2020).

The L-EG and gcm+ lineages undergo proliferation at the early pupal stage to generate the architecture of neuropil-glia in the adult, which is more complex and has 100-fold more glial cells than the larva. Adult flies process a vast amount of sensory information and exhibit complex behaviors, such as courtship, aggression, flight and walking. Accordingly, the structure of the adult brain is more elaborate, with more subdivided neuropils and 20-fold more neurons than the larval brain. Thus, the cell proliferation of both lineages leads to an increase in the number of glial cells, which likely occurs in coordination with the elaboration of adult neural circuits (Kato, 2020).

Neuron-glia interactions underlie the adjustment of glial cell numbers to neuronal structure through cell survival or cell proliferation in flies and vertebrates. This study shows that the FGF receptor htl is required for cell proliferation in both L-EG and gcm+ lineages in early pupal life. In flies, the htl ligand Pyramus, which is secreted from neurons, regulates the proliferation of htl-positive cortex glia during the larval stage (Avet-Rochex, 2012). Similarly, it is possible that htl ligands from neurons non-cell autonomously regulate the proliferation of lineage cells. Consistent with this notion, the data show that the total number of neuropil-glia in an area is limited, unless htl is constitutively activated. Thus, such non-cell autonomous regulation may adjust the numbers of neuropil-glia in adult neural circuits, thereby enabling the complex behavior of adult animals (Kato, 2020).

ALG and EG were present in both larval and adult brains, and each cell type shares morphological features and the expression of certain markers between stages. The data show the similarities in the developmental program of neuropil-glia for embryos/larvae and adults. It was ascertain that Pros is required for the differentiation of adult ALG, as it is for the development of embryonic/larval ALG. In embryos and larvae, Pros is also required to maintain the proliferative ability of ALG. The KD of pros in the lineages results in fewer GFP+ neuropil-glia at the 50% pupa stage in some areas of the brain. This implies that Pros is involved in the regulation of cell number in the development of adult neuropil-glia. The KD of pros in the cell lineages results in the appearance of Naz+ cells. The exact identity of these cells remains unknown. Rather than differentiating into adult ALG, the persisting weak Naz+ cells in adult brains may have acquired EG-like characteristics. However, it is difficult to assess this possibility because of the lack of markers that can clearly identify adult EG. Alternatively, they may be undifferentiated cells that have failed to differentiate into adult ALG. This notion is consistent with the fact that EG are Naz- and the undifferentiated cells at the 25% pupa stage are weak/faint Naz+ cells (Kato, 2020).

This study has established that htl is required for the proliferation of the L-EG and gcm+ lineages. In the development of embryonic/larval neuropil-glia, the number of ALG in htlAB42 null mutants is similar to that in the control, suggesting that htl is not involved in cell proliferation in embryos. Instead, htl is required for the proper organization of IG/ALG during embryogenesis, and for extending the fine projections of larval ALG into the neuropil. In the current analysis, the cell-proliferation phenotype of htl KD emerged in the early pupal stage; thus, the role of htl in later stages was not specifically investigated. htl is also expressed in neuropil-glia at the 50% pupa stage, when their maturation is initiated. Thus, these results do not rule out the involvement of htl in the maturation of adult ALG in later pupal stages. Nevertheless, they show that the developmental programs for larva and adult neuropil-glia partially differ (Kato, 2020).

The plasticity of glial cells in terms of their differentiation is well established in vertebrates. Radial glial progenitors generate neurons and, subsequently, some of them generate oligodendrocytes and astrocytes during development. Radial glia progenitors persist in adults and transform into neural stem cells. Both astrocytes and NG2-glia [oligodendrocyte precursor cells (OPCs)] in adult brains proliferate after injury, and generate astrocytes and oligodendrocytes, respectively. In some cases, astrocytes may even transdifferentiate into neurons after injury. NG2-glia/OPCs also generate astrocytes in cell culture, in development and after injury. Some studies also reported that NG2 glia generate neurons in adult mice. Drosophila ALG also proliferate in response to injury in larval ventral nerve cords. However, whether they differentiate into different glial subtypes or neurons is currently unexplored. Foo (2017a) reported the presence of adult neural progenitor cells in Drosophila that can generate glial cells and neurons in response to a defect in glial cells. In contrast, the changes of L-EG into the progenitor state, in which cells proliferate and then differentiate, are developmentally regulated. Thus, it may serve as an excellent model for the investigation of glial cell plasticity (Kato, 2020).

This study showed that adult neuropil-glia are derived from two lineages: L-EG and gcm+. What is the significance of having two lineages to establish the architecture of adult neuropil-glia? The peculiar distribution patterns of the lineages may relate to the evolution of insect brains. In numerous hemimetabolous insects (e.g. locusts and cockroaches), the mandibular, maxillary and labial ganglia, which are mostly occupied by L-EG lineage cells in flies, are detached from the protocerebrum, deutocerebrum and tritocerebrum, and located more inferiorly to (i.e., below) the esophagus. In these insects, L-EG may generate neuropil-glia for the mandibular, maxillary and labial ganglia, whereas gcm+ cells may generate neuropil-glia for the protocerebrum, deutocerebrum and tritocerebrum. In contrast, in flies, the two different populations appear to generate neuropil-glia for one structure (i.e. a brain) as all of the areas are fused together. This notion is consistent with the idea that the segmental distribution pattern of specific embryonic neuroblasts is evolutionarily conserved between Drosophila and hemimetabolous insects (Kato, 2020).

This study demonstrates that inhibition of glial proliferation in one lineage is rescued by the other lineage. This indicates that, regardless of evolutionary relevance, the multiple lineages (i.e., L-EG and gcm+ cells) ensure robust development of the adult neuropil-glia architecture. Such robust development of glial architecture has been reported in several contexts. In the thorax and brain, neuroblasts generate adult neuropil-glia and compensate for the failure of gliogenesis from other neuroblasts. This study reveals that the ability to compensate for deficiencies in a lineage is not restricted to neuroblast-derived glia and is greater in scope. Each lineage (L-EG or gcm+) rescues the entire loss of the other lineage. A similar mechanism is involved in the development of mouse oligodendrocytes. Two lineages of oligodendrocyte precursor cells (a ventral and a dorsal population) generate oligodendrocytes in embryos and in postnatal animals. One lineage completely takes over the brain when the other fails to develop, preventing any locomotor defect. Therefore, multiple lineages with glial ability to adjust to the surroundings guarantee the robust development of glial architecture. Thus, it is suggested that glial plasticity may be a widespread strategy for ensuring the robust development of functional brains (Kato, 2020).

Lineage-guided Notch-dependent gliogenesis by Drosophila multi-potent progenitors

Macroglial cells in the central nervous system exhibit regional specialization and carry out region-specific functions. Diverse glial cells arise from specific progenitors in specific spatiotemporal patterns. This raises an interesting possibility that there exist glial precursors with distinct developmental fates, which govern region-specific gliogenesis. This study mapped the glial progeny produced by the Drosophila type II neuroblasts, which, like vertebrate radial glia cells, yield both neurons and glia via intermediate neural progenitors (INPs). Distinct type II neuroblasts produce different characteristic sets of glia. A single INP can make both astrocyte-like and ensheathing glia, which co-occupy a relatively restrictive subdomain. Blocking apoptosis uncovers further lineage distinctions in the specification, proliferation, and survival of glial precursors. Both the switch from neurogenesis to gliogenesis and the subsequent glial expansion depend on Notch signaling. Taken together, lineage origins preconfigure the development of individual glial precursors with involvement of serial Notch actions in promoting gliogenesis (Ren, 2018).

The sulfite oxidase Shopper controls neuronal activity by regulating glutamate homeostasis in Drosophila ensheathing glia

Specialized glial subtypes provide support to developing and functioning neural networks. Astrocytes modulate information processing by neurotransmitter recycling and release of neuromodulatory substances, whereas ensheathing glial cells have not been associated with neuromodulatory functions yet. To decipher a possible role of ensheathing glia in neuronal information processing, a screen was carried out for glial genes required in the Drosophila central nervous system for normal locomotor behavior. Shopper (Sulfite oxidase) encodes a mitochondrial sulfite oxidase that is specifically required in ensheathing glia to regulate head bending and peristalsis. shopper mutants show elevated sulfite levels affecting the glutamate homeostasis which then act on neuronal network function. Interestingly, human patients lacking the Shopper homolog SUOX develop neurological symptoms, including seizures. Given an enhanced expression of SUOX by oligodendrocytes, the current findings might indicate that in both invertebrates and vertebrates more than one glial cell type may be involved in modulating neuronal activity (Otto, 2018).

Defective cortex glia plasma membrane structure underlies light-induced epilepsy in cpes mutants

Seizures induced by visual stimulation (photosensitive epilepsy; PSE) represent a common type of epilepsy in humans, but the molecular mechanisms and genetic drivers underlying PSE remain unknown, and no good genetic animal models have been identified as yet. This study shows an animal model of PSE, in Drosophila, owing to defective cortex glia. The cortex glial membranes are severely compromised in ceramide phosphoethanolamine synthase (cpes)-null mutants and fail to encapsulate the neuronal cell bodies in the Drosophila neuronal cortex. Expression of human sphingomyelin synthase 1, which synthesizes the closely related ceramide phosphocholine (sphingomyelin), rescues the cortex glial abnormalities and PSE, underscoring the evolutionarily conserved role of these lipids in glial membranes. Further, this study shows the compromise in plasma membrane structure that underlies the glial cell membrane collapse in cpes mutants and leads to the PSE phenotype (Kunduri, 2018).

Glial Ca(2+) signaling links endocytosis to K(+) buffering around neuronal somas to regulate excitability

Glial-neuronal signaling at synapses is widely studied, but how glia interact with neuronal somas to regulate neuronal function is unclear. Drosophila cortex glia are restricted to brain regions devoid of synapses, providing an opportunity to characterize interactions with neuronal somas. Mutations in the cortex glial NCKX(zydeco) elevate basal Ca(2+), predisposing animals to seizure-like behavior. To determine how cortex glial Ca(2+) signaling controls neuronal excitability, an in-vivo modifier screen of the NCKX(zydeco) seizure phenotype was performed. This study showe that elevation of glial Ca(2+) causes hyperactivation of calcineurin-dependent endocytosis and accumulation of early endosomes. Knockdown of sandman, a K2P channel, recapitulates NCKX(zydeco) seizures. Indeed, sandman expression on cortex glial membranes is substantially reduced in NCKX(zydeco) mutants, indicating enhanced internalization of Sandman predisposes animals to seizures. These data provide an unexpected link between glial Ca(2+) signaling and the well-known role of glia in K(+) buffering as a key mechanism for regulating neuronal excitability (Weiss, 2019).

Inhibiting glutamate activity during consolidation suppresses age-related long-term memory impairment in Drosophila

In Drosophila, long-term memory (LTM) formation requires increases in glial gene expression. Klingon (Klg), a cell adhesion molecule expressed in both neurons and glia, induces expression of the glial transcription factor, Repo. However, glial signaling downstream of Repo has been unclear. This study demonstrates that Repo increases expression of the glutamate transporter, EAAT1, and EAAT1 is required during consolidation of LTM. The expressions of Klg, Repo, and EAAT1 decrease upon aging, suggesting that age-related impairments in LTM are caused by dysfunction of the Klg-Repo-EAAT1 pathway. Supporting this idea, overexpression of Repo or EAAT1 rescues age-associated impairments in LTM. Pharmacological inhibition of glutamate activity during consolidation improves LTM in klg mutants and aged flies. Altogether, the results indicate that LTM formation requires glial-dependent inhibition of glutamate signaling during memory consolidation, and aging disrupts this process by inhibiting the Klg-Repo-EAAT1 pathway (Matsuno, 2019).

Changes in glial transcription due to neuronal activity have been studied previously, but a specific role of glial transcription in LTM has been less characterized. Expression of the glial transcription factor, Repo, increases shortly after spaced training, and this increase is required for LTM formation. This report has identified Eaat1 as a Repo-regulated glial gene required for LTM consolidation. Eaat1 encodes a glial glutamate transporter that removes glutamate from synaptic sites and transports it into astrocytes. Thus, the data indicate that glutamate signaling needs to be inhibited during LTM consolidation (Matsuno, 2019).

To identify Eaat1, a screen was performed for various genes regulating glial physiology for altered expression during LTM formation. Expression of Eaat1 and crammer was found to increase after spaced training. As Eaat1, but not crammer, is expressed exclusively in glia, focus was placed on Eaat1 as a likely Repo-regulated gene. Indeed, spaced-training-induced increases in EAAT1 depend on Repo and Klg activity. Interestingly, expression of the glial gene, genderblind, which encodes another glial glutamate transporter, required Repo activity for expression, but was not affected by spaced training, suggesting that other transcriptional regulatory factors besides Repo are likely necessary to differentially regulate genes required for memory consolidation from those required for other glial functions (Matsuno, 2019).

Because only screened selected genes were screened, it is possible that Repo induces the expression of other unidentified genes after spaced training. However, somewhat unexpectedly, it was found that overexpression of Eaat1 alone in glial cells is sufficient to rescue the LTM defects of klg and repo mutants. This indicates that the major function of the Klg/Repo signaling pathway is to induce glial expression of Eaat1. It further suggests that one function of astrocytes is to decrease glutamate signaling during LTM consolidation (Matsuno, 2019).

Combined with results from previous studies, this work identifies a putative pathway linking neuronal activity to glial inhibition of glutamate signaling. In flies, the homophilic cell adhesion molecule, Klingon, is expressed in both neurons and glia, and needs to be expressed in both cell types for normal LTM (Matsuno, 2015). Repo expression normally increases after spaced training, whereas it fails to do so in klg mutants, indicating that Klg-mediated neuron-glia communication is necessary for this increase (Matsuno, 2015). Thus, it is proposed that spaced training increases neuronal activity, which induces signaling to glia via the cell adhesion molecule Klg. This results in increased Repo activity in glia, which increases Eaat1 expression, and subsequently decreases glutamate signaling (Matsuno, 2019).

Previous work from various groups including has shown that glutamate signaling through NMDA-type receptors (NRs) is necessary for learning and memory. Overexpression of NRs in mice enhances learning and memory formation, and it has been shown that glial production of D-serine, a neuromodulator that functions as a coactivator of NRs, is necessary for short-lasting memory. In the current study, focus was placed on glutamate activity specifically during memory consolidation, instead of during initial learning and memory formation. Considering the current findings with those of previous studies, it is proposed that NR-dependent glutamate signaling needs to be initially high, during formation of short-lasting memories, but low during a later phase where short-lasting memories are consolidated into LTM. This suggests that glia play at least two roles in memory. They produce D-serine that contributes to high NR activity during memory formation and also produce EAAT1 after learning, which functions to reduce glutamate signaling during memory consolidation (Matsuno, 2019).

Age-related impairments in Drosophila memory do not consist of a general decrease in all forms of learning and memory, but instead consist of decreases in two specific phases of memory, MTM and LTM. The current results suggest that both these memory effects are caused by age-related glial dysfunction. Glia in young flies are able to produce sufficient amounts of D-serine for normal MTM, whereas D-serine amounts decrease 2-fold in aged flies. This decrease is responsible for age-related impairments in 1-h memory, because increasing glial production of D-serine, or directly feeding of D-serine to aged flies, rescues this impairment. Likewise, glial dysfunction is also responsible for age-related impairments in LTM because aged glia are unable to inhibit glutamate signaling during consolidation. Thus, in contrast to young flies, aged flies are unable to modulate glutamate activity during learning and consolidation, leading to defects in the two memory phases (Matsuno, 2019).

The model that EAAT1 inhibits glutamate activity during consolidation stems from EAAT1's role in clearing glutamate from synaptic sites and transporting it into astrocytes. This model is consistent with several mammalian studies that demonstrated decreased expression of astrocytic glutamate transporters upon aging, with a consequent reduction of glutamate uptake. Further supporting this model, it was found that feeding flies memantine or MK801, NMDA receptor antagonists, after spaced training, restores normal LTM in klg mutants and restores LTM in aged flies to youthful levels. This effect requires feeding after training during the consolidation phase. Similar results were obtained by feeding riluzole, a glutamate modulator, which decreases glutamate release and increases astrocytic glutamate uptake. Riluzole has also been reported to ameliorate age-related cognitive decline in mammals, suggesting that the mechanisms of AMI may be conserved between species. In contrast, this study found that D-serine feeding, which rescues age-related declines in short-lasting (1-h) memory, does not improve declines in LTM, but rather attenuates it. This is consistent with the model wherein declines in short-lasting memory and LTM are caused by distinct or opposing mechanisms and glutamate signaling needs to be suppressed during consolidation. Somewhat unexpectedly, it was also found that (s)-4C3HPG, the mGluR1 antagonist/mGluR2 agonist, also ameliorated age-related impairments in LTM. This result indicates that glutamate activity through both ionotropic and metabotropic glutamate receptors antagonizes memory consolidation (Matsuno, 2019).

Currently, it is unclear why glutamate signaling needs to be inhibited during consolidation, but a previous study has shown that Mg2+ block mutations in NMDA-type glutamate receptors (NRs) cause specific defects in LTM in Drosophila. Although Mg2+ block mutations have various effects, one effect is to increase NR activity. Increased NR activity results in increased activity of dCREB2b, an inhibitory isoform of CREB. CREB-dependent gene expression is required during consolidation of LTM, suggesting that consolidation may be preferentially sensitive to NR activity (Matsuno, 2019).

Alternatively, it is possible that neuronal activity needs to be inhibited globally during memory consolidation. Sleep is known to be important for LTM. Sleep deprivation during consolidation prevents LTM formation, whereas artificially inducing sleep after training has been reported to improve LTM. Thus a second possibility is suggested that inhibition of glutamate signaling after spaced training may be a brain-wide phenomenon that promotes consolidation by inducing the organism to sleep. Thus far, gross alterations in sleep duration in klg and repo mutants have not been detected, although this does not preclude minor disruptions in sleep quality that may not be detectable by motion-based sleep assays. Finally, a third possibility is envisioned wherein neuronal inhibition may be required as a neuroprotective mechanism that may be necessary to prevent cell death in neurons that were extensively stimulated during spaced training (Matsuno, 2019).

Mapping the glutamatergic neurons whose activity is inhibited during consolidation will be of great interest in the future. As aversive olfactory memories are formed and stored in the Drosophila MBs, it is possible that specific glutamatergic MB output neurons (MBONs) are inhibited during consolidation. Several glutamatergic MBONs are involved in feedback networks with the lobes of the MBs, suggesting that altering the activity of these neurons may modulate memory consolidation and memory-associated behavioral responses (Matsuno, 2019).

This study has demonstrate that increased expression of Eaat1 is required for LTM consolidation. Based on numerous results from other groups, it is hypothesized that Eaat1 functions to reduce glutamate signaling, and support for this model is provided by demonstrating that pharmacological inhibition of glutamate signaling during consolidation improves LTM under various conditions. However, due to technical limitations, it was not possible to actually measure glutamate concentrations at synapses during memory consolidation and it is not known where and how much glutamate signaling has to be inhibited for optimal LTM consolidation (Matsuno, 2019).

The pleiotropic effects of Innexin genes expressed in Drosophila glia encompass wing chemosensory sensilla

The neuroanatomy of Drosophila wing chemosensilla and the analysis of their sensory organ precursor cell lineage have demonstrated that they are surprisingly related to taste perception. The microarchitecture of wing bristles limits the use of electrophysiology methods to investigate wing chemosensory mechanisms. However, by monitoring the fluorescence of the complex calcium/GCaMP, calcium flux triggered upon tastant stimulation was observed within sensilla aligned along the wing anterior nerve. This string of fluorescent puncta was impaired in wings of Innexin 2 (Inx2) mutant flies; although it is unclear whether the Innexin proteins act at the level of the wing imaginal disc, adult wing and/or at both levels. Glial cells known to shelter Innexin(s) expression have no documented role in adult chemosensory sensilla. These data suggest that Innexin(s) are likely required for the maturation of functional wing chemosensilla in adulthood. The unexpected presence of most Innexin transcripts in adult wing RNAseq data set argues for the expression of Innexin proteins in the larval imaginal wing disc that are continued in wing chemosensilla at adulthood (Raad, 2019).

Neuronal lactate levels depend on glia-derived lactate during high brain activity in Drosophila

Lactate/pyruvate transport between glial cells and neurons is thought to play an important role in how brain cells sustain the high-energy demand that neuronal activity requires. However, the in vivo mechanisms and characteristics that underlie the transport of monocarboxylates are poorly described. This study used Drosophila expressing genetically encoded FRET sensors to provide an ex vivo characterization of the transport of monocarboxylates in motor neurons and glial cells from the larval ventral nerve cord. Lactate/pyruvate transport in glial cells was shown to be coupled to protons and is more efficient than in neurons. Glial cells maintain higher levels of intracellular lactate generating a positive gradient toward neurons. Interestingly, during high neuronal activity, raised lactate in motor neurons is dependent on transfer from glial cells mediated in part by the previously described monocarboxylate transporter Chaski, providing support for in vivo glia-to-neuron lactate shuttling during neuronal activity (Gonzalez-Gutierrez, 2019).

Li, H., Russo, A. and DiAntonio, A. (2019). SIK suppresses neuronal hyperexcitability by regulating the glial capacity to buffer K(+) and water. J Cell Biol 218(12):4017-4029. PubMed ID: 31645458

SIK suppresses neuronal hyperexcitability by regulating the glial capacity to buffer K(+) and water

Glial regulation of extracellular potassium (K(+)) helps to maintain appropriate levels of neuronal excitability. While channels and transporters mediating K(+) and water transport are known, little is understood about upstream regulatory mechanisms controlling the glial capacity to buffer K(+) and osmotically obliged water. This study identified salt-inducible kinase (SIK3) as the central node in a signal transduction pathway controlling glial K(+) and water homeostasis in Drosophila. Loss of SIK leads to dramatic extracellular fluid accumulation in nerves, neuronal hyperexcitability, and seizures. SIK3-dependent phenotypes are exacerbated by K(+) stress. SIK promotes the cytosolic localization of HDAC4, thereby relieving inhibition of Mef2-dependent transcription of K(+) and water transport molecules. This transcriptional program controls the glial capacity to regulate K(+) and water homeostasis and modulate neuronal excitability. HDAC was identified as a candidate therapeutic target in this pathway, whose inhibition can enhance the K(+) buffering capacity of glia, which may be useful in diseases of dysregulated K(+) homeostasis and hyperexcitability (Li, 2019).

Neuronal excitability is tightly coupled to complex ion dynamics in the nervous system. As ions move, so too must osmotically obliged water molecules move. Following bursts of action potentials, ionic gradients are restored via active transport. Defects in water and ion homeostasis disrupt neuronal firing and can result in edema. Potassium (K+) homeostasis is particularly important for maintaining the physiological function of neurons. Repolarization of the axonal membrane during action potentials transfers K+ ions into the extracellular space. With synchronous activity, the bulk extracellular [K+] rises, and this K+ must be rapidly buffered via glial and/or neurons lest axons depolarize, disrupting neuronal firing (Li, 2019).

Glial cells are essential for potassium and water homeostasis. Glia remove K+ ions from the extracellular space and redistribute them to areas with lower [K+] to maintain ionic gradients. Glia also express aquaporin water channels to functionally couple K+ clearance and water transport, thereby relieving osmotic stress. A number of K+ and water transport molecules mediate glial regulation of extracellular [K+], including K+ channels, Na+-K+-Cl−cotransporter 1, Na+-K+-ATPase, and aquaporin. Defects in glial transporters can result in build-up of ions in the extracellular space between axons and glia, leading to extracellular fluid accumulation. Indeed, mouse and Drosophila melanogaster mutants with defective K+transport exhibit dramatic and strikingly similar edema phenotypes in their peripheral nerves. These swellings correlate with seizure sensitivity and offer an elegant readout for identifying molecules that are required to maintain ionic and water homeostasis and a healthy level of neuronal excitability (Li, 2019).

While much is known about the key transporters and channels that mediate the flux of water and ions, the mechanisms by which glial cells regulate expression of the relevant transporters are not well understood. Delineating such regulatory mechanisms could identify approaches to leverage glial K+ and water buffering as a therapeutic strategy for neuroprotection against K+ stress-related damage. A glial-specific screen was conducted for this study in Drosophila, and a signal transduction pathway required for glial regulation of water and ion homeostasis was identified. This screen uncovered a central role for salt-inducible kinase (SIK3), a highly conserved AMP-activated protein kinase (AMPK)-family kinase that links signal sensing to changes in cellular response. Loss of SIK3 in glia results in nerve edema, neuronal hyperexcitability, and increased seizure susceptibility, all phenotypes that are commonly associated with human genetic disorders disrupting glial water and ion homeostasis. This swelling phenotype is critically and selectively sensitive to K+ stress. Moreover, this study demonstrates that SIK functions via regulation of a downstream HDAC4/Mef2 transcriptional program that controls expression of relevant ion and water transporters. HDAC is a critical negative regulator in the pathway, and pharmacological inhibition of HDAC potently suppresses the edema, hyperexcitability, and seizure phenotypes of SIK mutants. Hence, this study identifies a druggable pathway controlling the glial capacity to buffer K+ and water and a candidate therapeutic approach to achieve the long-standing goal of targeting glia for the control of hyperexcitability (Li, 2019).

Glia remove K+ from the extracellular space to maintain ionic and water homeostasis in the nervous system. Disrupting K+ buffering leads to nerve edema, neuronal hyperexcitability, and increased seizure susceptibility. In Drosophila, loss of glial K+ transport function results in characteristic focal peripheral nerve swellings due to accumulation of extracellular fluid. To identify signaling pathways required for glial regulation of K+ and water homeostasis, an in vivo glial-specific screen was performed in Drosophila. The pan-glial driver Repo-GAL was used to express a library of ~RNAi lines targeting signaling molecules, and were genes screened for that are required to prevent swelling of larval peripheral nerves. From the screen a single hit was found: glial knockdown of the gene for SIK3 results in pronounced nerve swellings (Li, 2019).

Glial wingless/Wnt regulates glutamate receptor clustering and synaptic physiology at the Drosophila neuromuscular junction

Glial cells are emerging as important regulators of synapse formation, maturation, and plasticity through the release of secreted signaling molecules. This study used chromatin immunoprecipitation along with Drosophila genomic tiling arrays to define potential targets of the glial transcription factor Reversed polarity (Repo). Unexpectedly, wingless (wg), encoding a secreted morphogen that regulates synaptic growth at the Drosophila larval neuromuscular junction (NMJ), was identified as a potential Repo target gene. Repo regulates wg expression in vivo, and local glial cells secrete Wg at the NMJ to regulate glutamate receptor clustering and synaptic function. This work identifies Wg as a novel in vivo glial-secreted factor that specifically modulates assembly of the postsynaptic signaling machinery at the Drosophila NMJ (Kerr, 2014).

The diversity of genes directly regulated by Repo-a critical transcriptional regulator of glial cell development in Drosophila-has not been thoroughly explored. ChIP studies from Drosophila S2 cells identified several potential Repo targets that have been shown to govern fundamental aspects of glial development or function. For example, known targets were identified that actively promote glial cell fate specification (e.g., pointed, distalless;, blood-brain barrier formation (e.g., gliotactin, loco, coracle, Nrv1, engulfment activity (e.g., dCed-6), neurotransmitter metabolism (e.g., EAAT1, Gs2), ionic homeostasis (e.g., fray), and neuron-glia signaling during nervous system morphogenesis (e.g., Pvr). For at least two potential targets, gs2 and Cp1, this study demonstrated a key requirement for Repo in their transcriptional activation during development (Kerr, 2014).

Given the broad roles of this collection of genes in glial cell biology, this work supports the hypothesis that Repo transcriptionally regulates a diverse class of genes that modulate many aspects of glial cell development. For instance, Pointed, which is now a predicted Repo target, is a key glial factor that activates glial fate at very early developmental stages. Likewise, Repo appears to regulate Gliotactin, Coracle, and Nrv1, which are molecules essential for formation of the pleated septate junction-based blood-brain barrier at mid to late embryogenesis in Drosophila. At the same time, EAAT1 and GS2 are activated late in the embryonic glial program, with expression being retained even in fully mature glia, and these transporters are critical for synaptic neurotransmitter recycling. Since EAAT1 and GS2 are both activated by Repo, and primarily expressed in CNS glia, these data argue that Repo is directly upstream of multiple key glial factors required for glutamate clearance from CNS synapses (Kerr, 2014).

Mammalian excitatory glutamatergic synapse formation is modulated by multiple soluble glia-derived factors including TSPs, Hevin/Sparc, and glypicans 4 and 6. These factors, along with other secreted glial factors that remain to be identified, are essential for initial synapse formation and (with the exception of TSPs) can promote postsynaptic differentiation through membrane insertion and clustering of AMPA receptors. This study identified Wg as a novel glia-derived factor essential for postsynaptic structure and function in vivo at the Drosophila glutamatergic NMJ. Combined with previous findings that NMJ glia can also release a TGF-β family member to regulate presynaptic growth in a retrograde manner (Fuentes-Medel, 2012), these studies provide compelling evidence that Drosophila glia function as a major integrator of synaptic signals during developmen (Kerr, 2014).

Previous work has demonstrated that Wg/Wnt signaling potently modulates the coordinated assembly of both presynaptic and postsynaptic structures at the Drosophila NMJ (Speese, 2007). Loss of Wg, or its receptor DFz2, leads to a dramatic decrease in synaptic boutons and disrupted clustering of postsynaptic glutamate receptors (Packard, 2002). Although previous studies supported evidence implicating motor neurons in Wg release, the presence of alternative cellular sources remained an open and important question. The surprising discovery of Wg as a candidate Repo target gene by ChIP led to an exploration of the possibility that NMJ glia could act as an additional in vivo source of NMJ Wg. Consistent with this idea, this study found that peripheral glia expressed Wg, SPGs were able to deliver Wg::GFP to the NMJ, and knockdown of SPG Wg significantly reduced NMJ Wg levels and led to a partial phenocopy of wg mutant phenotypes (Kerr, 2014).

Interestingly, it was found that loss of glia-derived Wg could account for some, but not all, wg loss-of-function phenotypes. For example, whereas depletion of glia-derived Wg disrupted clustering of postsynaptic glutamate receptors, it had no effect on the formation of synaptic boutons. In contrast, depletion of neuronal Wg led to defects in both glutamate receptor clustering as well as bouton formation. Although only neuronal Wg regulated bouton growth, these data argue that both glial and neuronal Wg are capable of modulating the assembly of glutamate receptor complexes. Thus, this study has identified two in vivo sources of Wg at the NMJ: the presynaptic neuron and local glial cells (Kerr, 2014).

Regarding the modulation of neurotransmission, both glial and neuronal Wg was found to have important roles, which, as in the case of the development of synaptic structure, were only partially overlapping. Loss of glial or neuronal Wg resulted in postsynaptic defects in neurotransmission, including increased mEJP amplitude (a postsynaptic property), decreased nerve-evoked EJPs, and decreased quantal content. Consistent with Repo regulating glial Wg expression, these phenotypes were mimicked by loss of repo function. The most notable difference in functional requirements for glial versus neuronal Wg is in mEJP frequency (a presynaptic function): depletion of glial Wg resulted in a dramatic increase in mEJP frequency, whereas manipulating neuronal Wg had no effect. Thus both glial and neuronal Wg are critical regulators of synaptic physiology in vivo, likely modulating NMJ neurotransmission in a combinatorial fashion, although glial Wg has the unique ability to modulate presynaptic function (Kerr, 2014).

The increase in mEJP amplitude is consistent with findings that GluR cluster size was increased upon loss of glia- or neuron-derived Wg, and that in general this was accompanied by minor changes in GluRIIA signal intensity. A potential explanation is that neuron- and glia-derived Wg regulate the levels of GluRIIA subunits. Previously, it was demonstrated that downregulation of the postsynaptic Frizzled Nuclear Import (FNI) pathway also increased GluRs at the NMJ (Speese, 2012). This suggests that glia- and neuron-derived Wg may act in concert via the FNI pathway to stabilize the synapse by regulating GluR expression (Kerr, 2014).

An important property of the larval NMJ is the ability to maintain constant synaptic function throughout development via structural and functional modifications. The combined functions of glial and neuronal Wg likely contribute to this mechanism, as together they positively regulate synaptic growth and function as well as organize postsynaptic machinery. However, a previous study suggested that the transcription factor Gooseberry (Gsb), in its role as positive regulator of synaptic homeostasis in neurons, may be antagonized by Wg function (Marie, 2010). Mutations in gsb block the increase in neurotransmitter release observed when postsynaptic GluRs are downregulated. Furthermore, Marie (2010) showed that the gsb mutant defect can be rescued by a heterozygous wg mutant allele. However, the specific role of Gsb in this process is unclear, as rapid synaptic homeostasis was normal in the mutant, and defects appeared restricted to a long-term decrease in GluR function. It will be important to define the specific role of Gsb in synaptic homeostasis and to manipulate Wg function in alternative ways before a clear relationship between Wg and Gsb can be established (Kerr, 2014).

How could neuronal versus glial Wg differ in regulating NMJ development and physiology? One possibility is that the level or site of Wg delivery by each cell type is different. For example, since SPGs invade the NMJ only intermittently (Fuentes-Medel, 2009), it is possible that they release most of their Wg outside of the NMJ, whereas the presynaptic neuron, which is embedded in the muscle cell, delivers it more efficiently and directly to the postsynaptic muscle cell. Alternatively, the Wg morphogen released by glia versus that released by neurons could be qualitatively different through alternative post-translational modifications such as glycosylation. Either mechanism would allow for glia to modulate specific aspects of NMJ physiology independently from neuronal Wg, perhaps in an activity-dependent manner (Kerr, 2014).

Although glia-derived Wg does not modulate NMJ growth, Drosophila glia can indeed regulate synaptic growth at the NMJ in vivo. It has been demonstrated previously that Drosophila glia release the TGF-β ligand Maverick to modulate TGF-β/BMP retrograde signaling at the NMJ and thereby the addition of new synaptic boutons (Fuentes-Medel, 2012). The discovery that glia-derived Wg can exert significant control over the physiological properties of NMJ synapses expands the mechanisms by which Drosophila glia can control NMJ synapse development and function. In the future it will be important to understand how glial Wg and TGF-β signaling integrate to promote normal NMJ growth, physiology, and plasticity (Kerr, 2014).

Glial response to hypoxia in mutants of NPAS1/3 homolog Trachealess through Wg signaling to modulate synaptic bouton organization

Synaptic structure and activity are sensitive to environmental alterations. Modulation of synaptic morphology and function is often induced by signals from glia. However, the process by which glia mediate synaptic responses to environmental perturbations such as hypoxia remains unknown. In the mutant for Trachealess (Trh), the Drosophila homolog for NPAS1 and NPAS3, smaller synaptic boutons form clusters named satellite boutons appear at larval neuromuscular junctions (NMJs), which is induced by the reduction of internal oxygen levels due to defective tracheal branches. Thus, the satellite bouton phenotype in the trh mutant is suppressed by hyperoxia, and recapitulated in wild-type larvae raised under hypoxia. It was further shown that hypoxia-inducible factor (HIF)-1alpha/Similar (Sima) is critical in mediating hypoxia-induced satellite bouton formation. Sima upregulates the level of the Wnt/Wingless (Wg) signal in glia, leading to reorganized microtubule structures within presynaptic sites. Finally, hypoxia-induced satellite boutons maintain normal synaptic transmission at the NMJs, which is crucial for coordinated larval locomotion (Chen, 2019).

genes expressed in glia


Aigouy, B., et al. (2004). Time-lapse and cell ablation reveal the role of cell interactions in fly glia migration and proliferation. Development 131: 5127-5138. PubMed ID: 15459105

Avet-Rochex, A., Kaul, A. K., Gatt, A. P., McNeill, H. and Bateman, J. M. (2012). Concerted control of gliogenesis by InR/TOR and FGF signalling in the Drosophila post-embryonic brain. Development 139(15): 2763-2772. PubMed ID: 22745312

Awasaki, T. and Ito, K. (2004). Engulfing action of glial cells is required for programmed axon pruning during Drosophila metamorphosis. Curr. Biol. 14: 668-677. PubMed ID: 15084281

Awasaki, T., Lai, S. L., Ito, K. and Lee, T. (2008). Organization and postembryonic development of glial cells in the adult central brain of Drosophila. J Neurosci 28(51): 13742-13753. PubMed ID: 19091965

Bainton, R. J. et al. (2005). moody encodes two GPCRs that regulate cocaine behaviors and blood-brain barrier permeability in Drosophila. Cell 123: 145-156. PubMed ID: 16213219

Bateman J. M. and McNeill H. (2004). Temporal control of differentiation by the insulin receptor/tor pathway in Drosophila. Cell 119: 87-96. PubMed ID: 15454083

Beckervordersandforth, R. M., Rickert, C., Altenhein, B. and Technau, C. M. (2008). Subtypes of glial cells in the Drosophila embryonic ventral nerve cord as related to lineage and gene expression. Mech. Dev. 125: 542-557. PubMed ID: 18296030

Bailey, A.P., Koster, G., Guillermier, C., Hirst, E.M., MacRae, J.I., Lechene, C.P., Postle, A.D. and Gould, A.P. (2015). Antioxidant role for lipid droplets in a stem cell niche of Drosophila. Cell 163: 340-353. PubMed ID: 26451484

Boulanger, A., Farge, M., Ramanoudjame, C., Wharton, K. and Dura, J. M. (2012). Drosophila motor neuron retraction during metamorphosis is mediated by inputs from TGF-beta/BMP signaling and orphan nuclear receptors. PLoS One 7: e40255. PubMed ID: 22792255

Charlton-Perkins, M. A., Sendler, E. D., Buschbeck, E. K. and Cook, T. A. (2017). Multifunctional glial support by Semper cells in the Drosophila retina. PLoS Genet 13(5): e1006782. PubMed ID: 28562601

Chen, P. Y., Tsai, Y. W., Cheng, Y. J., Giangrande, A. and Chien, C. T. (2019). Glial response to hypoxia in mutants of NPAS1/3 homolog Trachealess through Wg signaling to modulate synaptic bouton organization. PLoS Genet 15(8): e1007980. PubMed ID: 31381576

Chung, H. L., Wangler, M. F., Marcogliese, P. C., Jo, J., Ravenscroft, T. A., Zuo, Z., Duraine, L., Sadeghzadeh, S., Li-Kroeger, D., Schmidt, R. E., Pestronk, A., Rosenfeld, J. A., Burrage, L., Herndon, M. J., Chen, S., Shillington, A., Vawter-Lee, M., Hopkin, R., Rodriguez-Smith, J., Henrickson, M., Lee, B., Moser, A. B., Jones, R. O., Watkins, P., Yoo, T., Mar, S., Choi, M., Bucelli, R. C., Yamamoto, S., Lee, H. K., Prada, C. E., Chae, J. H., Vogel, T. P. and Bellen, H. J. (2020). Loss- or gain-of-function mutations in ACOX1 cause axonal loss via different Mechanisms. Neuron. PubMed ID: 32169171

Condron, B. and Zinn, K. (1995). Activation of cAMP-dependent protein kinase triggers a glial-to-neuronal cell-fate switch in an insect neuroblast lineage. Curr. Biol. 5: 51-61. PubMed ID: 7535171

Coutinho-Budd, J. C., Sheehan, A. E. and Freeman, M. R. (2017). The secreted neurotrophin Spatzle 3 promotes glial morphogenesis and supports neuronal survival and function. Genes Dev 31(20): 2023-2038. PubMed ID: 29138279

Daneman, T. and Barres, B. A. (2005). The blood-brain barrier -- lessons from moody flies. Cell 123: 9-12. PubMed ID: 16213208

Dearborn, R. and Kunes, S. (2004). An axon scaffold induced by retinal axons directs glia to destinations in the Drosophila optic lobe. Development 131: 2291-2303. PubMed ID: 15102705

De Graeve, F., et al. (2004). The ladybird homeobox genes are essential for the specification of a subpopulation of neural cells. Dev. Biol. 270: 122-134. PubMed ID: 15136145

Delgado, M. G., Oliva, C., Lopez, E., Ibacache, A., Galaz, A., Delgado, R., Barros, L. F. and Sierralta, J. (2018). Chaski, a novel Drosophila lactate/pyruvate transporter required in glia cells for survival under nutritional stress. Sci Rep 8(1): 1186. PubMed ID: 29352169

Di Cara, F., Rachubinski, R. A. and Simmonds, A. J. (2019). Distinct roles for peroxisomal targeting signal receptors Pex5 and Pex7 in Drosophila. Genetics 211(1): 141-149. PubMed ID: 30389805

Doherty, J., Logan, M. A., Tasdemir, O. E. and Freeman, M. R. (2009). Ensheathing glia function as phagocytes in the adult Drosophila brain. J. Neurosci. 29(15): 4768-81. PubMed ID: 19369546

Dourlen, P., Bertin, B., Chatelain, G., Robin, M., Napoletano, F., Roux, M. J. and Mollereau, B. (2012). Drosophila fatty acid transport protein regulates rhodopsin-1 metabolism and is required for photoreceptor neuron survival. PLoS Genet 8(7): e1002833. PubMed ID: 22844251

Enriquez, J., Rio, L. Q., Blazeski, R., Bellemin, S., Godement, P., Mason, C. and Mann, R. S. (2018). Differing strategies despite shared lineages of motor neurons and glia to achieve robust development of an adult neuropil in Drosophila. Neuron 97(3): 538-554.e535. PubMed ID: 29395908

Fischbach, K.-F. and Technau, G. (1984). Cell degeneration in the developing optic lobes of the small optic lobes and sine oculis mutants of Drosophila melanogaster. Dev. Biol. 104: 219-239. PubMed ID: 6428950

Foo, L. C., Song, S. and Cohen, S. M. (2017a). miR-31 mutants reveal continuous glial homeostasis in the adult Drosophila brain. EMBO J 36(9):1215-1226. PubMed ID: 28320737

Foo, L. C. (2017b). Cyclin-dependent kinase 9 is required for the survival of adult Drosophila melanogaster glia. Sci Rep 7(1): 6796. PubMed ID: 28754981

Fuentes-Medel, Y., Logan, M. A., Ashley, J., Ataman, B., Budnik, V. and Freeman, M. R. (2009). Glia and muscle sculpt neuromuscular arbors by engulfing destabilized synaptic boutons and shed presynaptic debris. PLoS Biol 7(8): e1000184. PubMed ID: 19707574

Fuentes-Medel, Y., Ashley, J., Barria, R., Maloney, R., Freeman, M. and Budnik, V. (2012). Integration of a retrograde signal during synapse formation by glia-secreted TGF-beta ligand. Curr Biol 22: 1831-1838. PubMed ID: 22959350

Gerdoe-Kristensen, S., Lund, V. K., Wandall, H. H. and Kjaerulff, O. (2016). Mactosylceramide prevents glial cell overgrowth by inhibiting insulin and fibroblast growth factor receptor signaling. J Cell Physiol [Epub ahead of print]. PubMed ID: 28019653

Ghosh, A., Kling, T., Snaidero, N., Sampaio, J. L., Shevchenko, A., Gras, H., Geurten, B., Gopfert, M. C., Schulz, J. B., Voigt, A. and Simons, M. (2013). A global in vivo Drosophila RNAi screen identifies a key role of ceramide phosphoethanolamine for glial ensheathment of axons. PLoS Genet 9(12): e1003980. PubMed ID: 24348263

Gonzalez-Gutierrez, A., Ibacache, A., Esparza, A., Barros, L. F. and Sierralta, J. (2019). Neuronal lactate levels depend on glia-derived lactate during high brain activity in Drosophila. Glia. PubMed ID: 31876077

Goncalves-Pimentel, C., Mazaud, D., Kottler, B., Proelss, S., Hirth, F. and Fanto, M. (2020). A miRNA screen procedure identifies garz as an essential factor in adult glia functions and validates Drosophila as a beneficial 3Rs model to study glial functions and GBF1 biology. F1000Res 9: 317. PubMed ID: 32595956

Gronke S., et al. Partridge L. (2010). Molecular evolution and functional characterization of Drosophila insulin-like peptides. PLoS Genet. 6: e1000857. PubMed ID: 20195512

Hakim, Y., Yaniv, S. P. and Schuldiner, O. (2014). Astrocytes play a key role in Drosophila mushroom body axon pruning. PLoS One 9: e86178. PubMed ID: 24465945

Hernandez, E., MacNamee, S. E., Kaplan, L. R., Lance, K., Garcia-Verdugo, H. D., Farhadi, D. S., Deer, C., Lee, S. W. and Oland, L. A. (2020). The astrocyte network in the ventral nerve cord neuropil of the Drosophila third-instar larva. J Comp Neurol. PubMed ID: 31909826

Hidalgo, A. and Booth, G. E. (2000). Glia dictate pioneer axon trajectories in the Drosophila embryonic CNS. Development 127: 393-402. PubMed ID: 10603355

Hoopfer, E. D., McLaughlin, T., Watts, R. J., Schuldiner, O., O'Leary, D. D. and Luo, L. (2006). Wlds protection distinguishes axon degeneration following injury from naturally occurring developmental pruning. Neuron 50(6): 883-95. PubMed ID: 16772170

Huang, T. H., Velho, T. and Lois, C. (2016). Monitoring cell-cell contacts in vivo in transgenic animals. Development 143: 4073-4084. PubMed ID: 27660327

Ito, K., Urban, J. and Technau, G. M. (1995). Distribution, classification and development of Drosophila glial cells in the late embryonic and early larval ventral nerve cord. Roux's Arch. Dev. Biol. 204: 284-307

Jones, B.W., Fetter, R.D., Tear, G. and Goodman, C.S. (1995). glial cells missing: a genetic switch that controls glial versus neuronal fate. Cell 82: 1013-1023. PubMed ID: 7553843

Kassmann, C. M. (2014). Myelin peroxisomes: essential organelles for the maintenance of white matter in the nervous system. Biochimie 98: 111-118. PubMed ID: 24120688

Kis, V., Barti, B., Lippai, M. and Sass, M. (2015). Specialized cortex glial cells accumulate lipid droplets in Drosophila melanogaster. PLoS One 10(7): e0131250. PubMed ID: 26148013

Kato, K., Orihara-Ono, M. and Awasaki, T. (2020). Multiple lineages enable robust development of the neuropil-glia architecture in adult Drosophila. Development 147(5). PubMed ID: 32051172

Kazama, H., Yaksi, E. and Wilson, R. I. (2011). Cell death triggers olfactory circuit plasticity via glial signaling in Drosophila. J. Neurosci. 31(21): 7619-30. PubMed ID: 21613475

Kerr, K. S., Fuentes-Medel, Y., Brewer, C., Barria, R., Ashley, J., Abruzzi, K. C., Sheehan, A., Tasdemir-Yilmaz, O. E., Freeman, M. R. and Budnik, V. (2014). Glial wingless/Wnt regulates glutamate receptor clustering and synaptic physiology at the Drosophila neuromuscular junction. J Neurosci 34: 2910-2920. PubMed ID: 24553932

Kremer, M. C., Jung, C., Batelli, S., Rubin, G. M. and Gaul, U. (2017). The glia of the adult Drosophila nervous system. Glia 65(4): 606-638. PubMed ID: 28133822

Kunduri, G., Turner-Evans, D., Konya, Y., Izumi, Y., Nagashima, K., Lockett, S., Holthuis, J., Bamba, T., Acharya, U. and Acharya, J. K. (2018). Defective cortex glia plasma membrane structure underlies light-induced epilepsy in cpes mutants. Proc Natl Acad Sci U S A 115(38): E8919-e8928. PubMed ID: 30185559

Larsen, P. H. and Yong, V. W. (2004). The expression of matrix metalloproteinase-12 by oligodendrocytes regulates their maturation and morphological differentiation. J. Neurosci. 24: 7597-7603. PubMed ID: 15342725

Larsen, P. H., et al. (2006). Myelin formation during development of the CNS is delayed in matrix metalloproteinase-9 and -12 null mice. J. Neurosci. 26: 2207-2214. PubMed ID: 16495447

Li, H., Russo, A. and DiAntonio, A. (2019). SIK suppresses neuronal hyperexcitability by regulating the glial capacity to buffer K(+) and water. J Cell Biol 218(12):4017-4029. PubMed ID: 31645458

Lin, G., Lee, P. T., Chen, K., Mao, D., Tan, K. L., Zuo, Z., Lin, W. W., Wang, L. and Bellen, H. J. (2018). Phospholipase PLA2G6, a Parkinsonism-associated gene, affects Vps26 and Vps35, retromer function, and ceramide levels, similar to alpha-Synuclein gain. Cell Metab 28(4): 605-618 e606. PubMed ID: 29909971

Liu, L., Zhang, K., Sandoval, H., Yamamoto, S., Jaiswal, M., Sanz, E., Li, Z., Hui, J., Graham, B. H., Quintana, A. and Bellen, H. J. (2015). Glial lipid droplets and ROS induced by mitochondrial defects promote neurodegeneration. Cell 160(1-2): 177-190. PubMed ID: 25594180

Liu, L., MacKenzie, K. R., Putluri, N., Maletic-Savatic, M. and Bellen, H. J. (2017). The glia-geuron lactate shuttle and elevated ROS promote lipid synthesis in neurons and lipid droplet accumulation in glia via APOE/D. Cell Metab 26(5): 719-737.e716. PubMed ID: 28965825

Lu, T. Y., Doherty, J. and Freeman, M. R. (2014). DRK/DOS/SOS converge with Crk/Mbc/dCed-12 to activate Rac1 during glial engulfment of axonal debris. Proc Natl Acad Sci U S A. PubMed ID: 25099352

Marie, B., Pym, E., Bergquist, S. and Davis, G. W. (2010). Synaptic homeostasis is consolidated by the cell fate gene gooseberry, a Drosophila pax3/7 homolog. J Neurosci 30: 8071-8082. PubMed ID: 20554858

Mast, F. D., Li, J., Virk, M. K., Hughes, S. C., Simmonds, A. J. and Rachubinski, R. A. (2011). A Drosophila model for the Zellweger spectrum of peroxisome biogenesis disorders. Dis Model Mech 4(5): 659-672. PubMed ID: 21669930

McHugh, B., et al. (2004). Invadolysin: a novel, conserved metalloprotease links mitotic structural rearrangements with cell migration. J. Cell Biol. 167: 673-686. PubMed ID: 15557119

MacNamee, S. E., Liu, K. E., Gerhard, S., Tran, C. T., Fetter, R. D., Cardona, A., Tolbert, L. P. and Oland, L. A. (2016). Astrocytic glutamate transport regulates a Drosophila CNS synapse that lacks astrocyte ensheathment. J Comp Neurol [Epub ahead of print]. PubMed ID: 27073064

Matsuno, M., Horiuchi, J., Tully, T. and Saitoe, M. (2009). The Drosophila cell adhesion molecule klingon is required for long-term memory formation and is regulated by Notch. Proc Natl Acad Sci U S A 106(1): 310-315. PubMed ID: 19104051

Matsuno, M., Horiuchi, J., Yuasa, Y., Ofusa, K., Miyashita, T., Masuda, T. and Saitoe, M. (2015). Long-term memory formation in Drosophila requires training-dependent glial transcription. J Neurosci 35(14): 5557-5565. PubMed ID: 25855172

Matsuno, M., Horiuchi, J., Ofusa, K., Masuda, T. and Saitoe, M. (2019). Inhibiting glutamate activity during consolidation suppresses age-related long-term memory impairment in Drosophila. iScience 15: 55-65. PubMed ID: 31030182

McNeill H., Craig G. M. and Bateman J. M. (2008). Regulation of neurogenesis and epidermal growth factor receptor signalling by the insulin receptor/target of rapamycin pathway in Drosophila. Genetics 179: 843-853. PubMed ID: 18505882

Nakano, R., Iwamura, M., Obikawa, A., Togane, Y., Hara, Y., Fukuhara, T., Tomaru, M., Takano-Shimizu, T. and Tsujimura, H. (2019). Cortex glia clear dead young neurons via Drpr/dCed-6/Shark and Crk/Mbc/dCed-12 signaling pathways in the developing Drosophila optic lobe. Dev Biol 453(1):68-85. PubMed ID: 31063730

Nelson, H. B. and Laughon, A. (1994) Drosophila glial development is regulated by genes involved in the control of neuronal cell fate. Roux's Arch. Dev. Biol. 204: 118-125

Ng, F. S., Sengupta, S., Huang, Y., Yu, A. M., You, S., Roberts, M. A., Iyer, L. K., Yang, Y. and Jackson, F. R. (2016). TRAP-seq profiling and RNAi-based genetic screens identify conserved glial genes required for adult Drosophila behavior. Front Mol Neurosci 9: 146. PubMed ID: 28066175

Otto, N., Marelja, Z., Schoofs, A., Kranenburg, H., Bittern, J., Yildirim, K., Berh, D., Bethke, M., Thomas, S., Rode, S., Risse, B., Jiang, X., Pankratz, M., Leimkuhler, S. and Klambt, C. (2018). The sulfite oxidase Shopper controls neuronal activity by regulating glutamate homeostasis in Drosophila ensheathing glia. Nat Commun 9(1): 3514. PubMed ID: 30158546

Ou, J., Gao, Z., Song, L. and Ho, M. S. (2016). Analysis of glial distribution in Drosophila adult brains. Neurosci Bull [Epub ahead of print]. PubMed ID: 26810782

Packard, M., Koo, E. S., Gorczyca, M., Sharpe, J., Cumberledge, S. and Budnik, V. (2002). The Drosophila Wnt, wingless, provides an essential signal for pre- and postsynaptic differentiation. Cell 111: 319-330. PubMed ID: 12419243

Petley-Ragan, L. M., Ardiel, E. L., Rankin, C. H. and Auld, V. J. (2016). Accumulation of laminin monomers in Drosophila glia leads to glial endoplasmic reticulum stress and disrupted larval locomotion. J Neurosci 36(4): 1151-1164. PubMed ID: 26818504

Raad, H. and Robichon, A. (2019). The pleiotropic effects of Innexin genes expressed in Drosophila glia encompass wing chemosensory sensilla. J Neurosci Res. PubMed ID: 31257643

Reddy, B. V. and Irvine, K. D. (2011). Regulation of Drosophila glial cell proliferation by Merlin-Hippo signaling. Development 138(23): 5201-12. PubMed ID: 22069188

Ren, Q., Awasaki, T., Wang, Y. C., Huang, Y. F. and Lee, T. (2018). Lineage-guided Notch-dependent gliogenesis by Drosophila multi-potent progenitors. Development. PubMed ID: 29764857

Rossi, A. M. and Fernandes, V. M. (2018). Wrapping glial morphogenesis and signaling control the timing and pattern of neuronal differentiation in the Drosophila lamina. J Exp Neurosci 12: 1179069518759294. PubMed ID: 29531474

Ryglewski, S., Duch, C. and Altenhein, B. (2017). Tyramine actions on Drosophila flight behavior are affected by a glial dehydrogenase/reductase. Front Syst Neurosci 11: 68. PubMed ID: 29021745

Sachse, S., et al. (2007). Activity-dependent plasticity in an olfactory circuit. Neuron 56: 838-850. PubMed ID: 18054860

Schwabe, T., et al. (2005). GPCR signaling is required for blood-brain barrier formation in Drosophila. Cell 123: 133-144. PubMed ID: 16213218

Schwabe, T., Li, X. and Gaul, U. (2017). Dynamic analysis of the mesenchymal-epithelial transition of blood-brain barrier forming glia in Drosophila. Biol Open 6(2):232-243. PubMed ID: 28108476

Sepp, K. J., Schulte, J. and Auld, V. J. (2000). Developmental dynamics of peripheral glia in Drosophila melanogaster. Glia 30: 122-133. PubMed ID: 10719354

Sen, A., Shetty, C., Jhaveri, D. and Rodrigues, V. (2005). Distinct types of glial cells populate the Drosophila antenna. BMC Dev. Biol. 5: 25. PubMed ID: 16281986

Sieglitz, F., Matzat, T., Yuva-Adyemir, Y., Neuert, H., Altenhein, B. and Klambt, C. (2013). Antagonistic feedback loops involving rau and sprouty in the Drosophila eye control neuronal and glial differentiation. Sci Signal 6: ra96. PubMed ID: 24194583

Speese, S. D. and Budnik, V. (2007). Wnts: up-and-coming at the synapse. Trends Neurosci 30: 268-275. PubMed ID: 17467065

Spindler, S. R., Ortiz, I., Fung, S., Takashima, S. and Hartenstein, V. (2009). Drosophila cortex and neuropile glia influence secondary axon tract growth, pathfinding, and fasciculation in the developing larval brain. Dev. Biol. 334(2): 355-68. PubMed ID: 19646433

Stork, T., et al. (2008). Organization and function of the blood-brain barrier in Drosophila. J. Neurosci. 28(3): 587-597. PubMed ID: 18199760

Stork, T., Sheehan, A., Tasdemir-Yilmaz, O. E., Freeman, M. R. (2014) Neuron-glia interactions through the Heartless FGF receptor signaling pathway mediate morphogenesis of Drosophila astrocytes. Neuron 83: 388-403. PubMed ID: 25033182

Subramanian, A., Siefert, M., Banerjee, S., Vishal, K., Bergmann, K. A., Curts, C. C. M., Dorr, M., Molina, C. and Fernandes, J. (2017). Remodeling of peripheral nerve ensheathment during the larval-to-adult transition in Drosophila. Dev Neurobiol 77(10): 1144-1160. PubMed ID: 28388016

Tasdemir-Yilmaz, O. E. and Freeman, M. R. (2013). Astrocytes engage unique molecular programs to engulf pruned neuronal debris from distinct subsets of neurons. Genes Dev. 28(1): 20-33. PubMed ID: 24361692

Unhavaithaya, Y. and Orr-Weaver, T. L. (2012). Polyploidization of glia in neural development links tissue growth to blood-brain barrier integrity. Genes Dev. 26(1): 31-6. PubMed ID: 22215808

Vasenkova, I., Luginbuhl, D. and Chiba, A. (2005). Gliopodia extend the range of direct glia-neuron communication during the CNS development in Drosophila. Mol. Cell. Neurosci. 31(1): 123-30. PubMed ID: 16298140

Viktorin, G., et al. (2011). Multipotent neural stem cells generate glial cells of the central complex through transit amplifying intermediate progenitors in Drosophila brain development. Dev. Biol. 356(2): 553-65. PubMed ID: 21708145

Volkenhoff, A., Weiler, A., Letzel, M., Stehling, M., Klambt, C. and Schirmeier, S. (2015). Glial glycolysis is essential for neuronal survival in Drosophila. Cell Metab [Epub ahead of print]. PubMed ID: 26235423

von Hilchen, C. M., et al. (2008). Identity, origin, and migration of peripheral glial cells in the Drosophila embryo. Mech. Dev. 125: 337-352. PubMed ID: 18077143

Wangler, M. F., Chao, Y. H., Bayat, V., Giagtzoglou, N., Shinde, A. B., Putluri, N., Coarfa, C., Donti, T., Graham, B. H., Faust, J. E., McNew, J. A., Moser, A., Sardiello, M., Baes, M. and Bellen, H. J. (2017). Peroxisomal biogenesis is genetically and biochemically linked to carbohydrate metabolism in Drosophila and mouse. PLoS Genet 13(6): e1006825. PubMed ID: 28640802

Weiss, S., Melom, J. E., Ormerod, K. G., Zhang, Y. V. and Littleton, J. T. (2019). Glial Ca(2+) signaling links endocytosis to K(+) buffering around neuronal somas to regulate excitability. Elife 8. PubMed ID: 31025939

Wheeler, S. R., Pearson, J. C. and Crews, S. T. (2012). Time-lapse imaging reveals stereotypical patterns of Drosophila midline glial migration. Dev. Biol. 361(2): 232-44. PubMed ID: 22061481

Wu, B., Li, J., Chou, Y. H., Luginbuhl, D. and Luo, L. (2017). Fibroblast growth factor signaling instructs ensheathing glia wrapping of Drosophila olfactory glomeruli. Proc Natl Acad Sci U S A. PubMed ID: 28674010

Xie, X., Gilbert, M., Petley-Ragan, L., Auld, V. J. (2014), Loss of focal adhesions in glia disrupts both glial and photoreceptor axon migration in the Drosophila visual system. Development 141: 3072-3083. PubMed ID: 25053436

Ye, Y., Gu, L., Chen, X., Shi, J., Zhang, X. and Jiang, C. (2016). Chromatin remodeling during the in vivo glial differentiation in early Drosophila embryos. Sci Rep 6: 33422. PubMed ID: 27634414

Ziegenfuss, J. S., Doherty, J. and Freeman, M. R. (2012). Distinct molecular pathways mediate glial activation and engulfment of axonal debris after axotomy. Nat Neurosci 15(7): 979-987. PubMed ID: 22706267

Zulbahar, S., Sieglitz, F., Kottmeier, R., Altenhein, B., Rumpf, S. and Klambt, C. (2018). Differential expression of the Drosophila Ntan/Obek controls ploidy in the blood-brain barrier. Development. PubMed ID: 30002129

Home page: The Interactive Fly © 1995, 1996 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.