The Interactive Fly
Genes involved in tissue and organ development
Genes influencing glial differentiation
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.
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). 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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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).
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) 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).
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): 2763-72. 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): 13742-53. 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
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
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
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
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
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
Gronke S., et al. Partridge L. (2010). Molecular evolution and functional characterization of Drosophila insulin-like peptides. PLoS Genet. 6: e1000857. PubMed ID: 20195512
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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