Glia engulf degenerating axons during developmental axon pruning

Developmental axon pruning is widely used in constructing the nervous system. Accordingly, diverse mechanisms are likely employed for various forms of axon pruning. In the Drosophila mushroom bodies (MB), gamma neurons initially extend axon branches into both the dorsal and medial MB axon lobes in larvae. Through a well-orchestrated set of developmental events during metamorphosis, axon branches to both lobes degenerate prior to the formation of adult connections. Here, we analyze ultrastructural changes underlying axon pruning by using a genetically encoded electron microscopic (EM) marker to selectively label gamma neurons. By inhibiting axon pruning in combination with the use of this EM marker, we demonstrate a causal link between observed cellular events and axon pruning. These events include changes in axon ultrastructure, synaptic degeneration, and engulfment of degenerating axon fragments by glia for their subsequent breakdown via the endosomal-lysosomal pathway. Interestingly, glia selectively invade MB axon lobes at the onset of metamorphosis; this increase in cell number is independent of axon fragmentation. Our study reveals a key role for glia in the removal of axon fragments during developmental axon pruning.     

Axon pruning during Drosophila metamorphosis: evidence for local degeneration and requirement of the ubiquitin-proteasome system

Axon pruning is widely used for the refinement of neural circuits in both vertebrates and invertebrates, and may also contribute to the pathogenesis of neurodegenerative diseases. However, little is known about the cellular and molecular mechanisms of axon pruning. We use the stereotyped pruning of gamma neurons of the Drosophila mushroom bodies (MB) during metamorphosis to investigate these mechanisms. Detailed time course analyses indicate that MB axon pruning is mediated by local degeneration rather than retraction and that the disruption of the microtubule cytoskeleton precedes axon pruning. In addition, multiple lines of genetic evidence demonstrate an intrinsic role of the ubiquitin-proteasome system in axon pruning; for example, loss-of-function mutations of the ubiquitin activating enzyme (E1) or proteasome subunits in MB neurons block axon pruning. Our findings suggest that some forms of axon pruning during development may share similarities with degeneration of axons in response to injury.     

Developmentally programmed remodeling of the Drosophila olfactory circuit

Neural circuits are often remodeled after initial connections are established. The mechanisms by which remodeling occurs, in particular whether and how synaptically connected neurons coordinate their reorganization, are poorly understood. In Drosophila, olfactory projection neurons (PNs) receive input by synapsing with olfactory receptor neurons in the antennal lobe and relay information to the mushroom body (MB) calyx and lateral horn. Here we show that embryonic-born PNs participate in both the larval and adult olfactory circuits. In the larva, these neurons generally innervate a single glomerulus in the antennal lobe and one or two glomerulus-like substructures in the MB calyx. They persist in the adult olfactory circuit and are prespecified by birth order to innervate a subset of glomeruli distinct from larval-born PNs. Developmental studies indicate that these neurons undergo stereotyped pruning of their dendrites and axon terminal branches locally during early metamorphosis. Electron microscopy analysis reveals that these PNs synapse with MB gamma neurons in the larval calyx and that these synaptic profiles are engulfed by glia during early metamorphosis. As with MB gamma neurons, PN pruning requires cell-autonomous reception of the nuclear hormone ecdysone. Thus, these synaptic partners are independently programmed to prune their dendrites and axons.     

Essential role of the apoptotic cell engulfment genes draper and ced-6 in programmed axon pruning during Drosophila metamorphosis

Axon pruning is a common phenomenon in neural circuit development. Previous studies demonstrate that the engulfing action of glial cells is essential in this process. The underlying molecular mechanisms, however, remain unknown. We show that draper (drpr) and ced-6, which are essential for the clearance of apoptotic cells in C. elegans, function in the glial engulfment of larval axons during Drosophila metamorphosis. The drpr mutation and glia-specific knockdown of drpr and ced-6 by RNA interference suppress glial engulfment, resulting in the inhibition of axon pruning. drpr and ced-6 interact genetically in the glial action. Disruption of the microtubule cytoskeleton in the axons to be pruned occurs via ecdysone signaling, independent of glial engulfment. These findings suggest that glial cells engulf degenerating axons through drpr and ced-6. We propose that apoptotic cells and degenerating axons of living neurons are removed by a similar molecular mechanism.     

The L1-CAM, Neuroglian, functions in glial cells for Drosophila antennal lobe development

Although considerable progress has been made in understanding the roles of olfactory receptor neurons (ORNs) and projection neurons (PNs) in Drosophila antennal lobe (AL) development, the roles of glia have remained largely mysterious. Here, we show that during Drosophila metamorphosis, a population of midline glial cells in the brain undergoes extensive cellular remodeling and is closely associated with the collateral branches of ORN axons. These glial cells are required for ORN axons to project across the midline and establish the contralateral wiring in the ALs. We find that Neuroglian (Nrg), the Drosophila homolog of the vertebrate cell adhesion molecule, L1, is expressed and functions in the midline glial cells to regulate their proper development. Loss of Nrg causes the disruption in glial morphology and the agenesis of the antennal commissural tract. Our genetic analysis further demonstrates that the functions of Nrg in the midline glia require its ankyrin-binding motif. We propose that Nrg is an important regulator of glial morphogenesis and axon guidance in AL development.     

Astrocytes Play a Key Role in Drosophila Mushroom Body Axon Pruning

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

The Drosophila cell corpse engulfment receptor Draper mediates glial clearance of severed axons

Neuron-glia communication is central to all nervous system responses to trauma, yet neural injury signaling pathways remain poorly understood. Here we explore cellular and molecular aspects of neural injury signaling in Drosophila. We show that transected Drosophila axons undergo injury-induced degeneration that is morphologically similar to Wallerian degeneration in mammals and can be suppressed by the neuroprotective mouse Wlds protein. Axonal injury elicits potent morphological and molecular responses from Drosophila glia: glia upregulate expression of the engulfment receptor Draper, undergo dramatic changes in morphology, and rapidly recruit cellular processes toward severed axons. In draper mutants, glia fail to respond morphologically to axon injury, and severed axons are not cleared from the CNS. Thus Draper appears to act as a glial receptor for severed axon-derived molecular cues that drive recruitment of glial processes to injured axons for engulfment.     

Cell-autonomous requirement of the USP/EcR-B ecdysone receptor for mushroom body neuronal remodeling in Drosophila

Neuronal process remodeling occurs widely in the construction of both invertebrate and vertebrate nervous systems. During Drosophila metamorphosis, gamma neurons of the mushroom bodies (MBs), the center for olfactory learning in insects, undergo pruning of larval-specific dendrites and axons followed by outgrowth of adult-specific processes. To elucidate the underlying molecular mechanisms, we conducted a genetic mosaic screen and identified one ultraspiracle (usp) allele defective in larval process pruning. Consistent with the notion that USP forms a heterodimer with the ecdysone receptor (EcR), we found that the EcR-B1 isoform is specifically expressed in the MB gamma neurons, and is required for the pruning of larval processes. Surprisingly, most identified primary EcR/USP targets are dispensable for MB neuronal remodeling. Our study demonstrates cell-autonomous roles for EcR/USP in controlling neuronal remodeling, potentially through novel downstream targets.     

Glial remodeling during metamorphosis influences the stabilization of motor neuron branches in Drosophila

Motor neurons that innervate the dorsal longitudinal (flight) muscles, DLMs, make multiple points of contact along the length of fibers. The stereotypy of the innervation lies in the number of contact points (CPs) made by each motor neuron and is established as a consequence of pruning that occurs during metamorphosis. Coincident with the onset of pruning is the arrival of glial processes that eventually ensheath persistent branches. To test a possible role for glia during pruning, the development of adult-specific glial ensheathment was disrupted using a targeted expression of dominant negative shibire. Such a manipulation resulted in fewer contact points at the DLM fibers. The development of innervation was examined during metamorphosis, specifically to test if the reduction was a consequence of increased pruning. We quantified the number of branches displaying discontinuities in their membrane, an indicator of the level of pruning. Disrupting the formation of glial ensheathment resulted in a two-fold increase in the discontinuities, indicating that pruning is enhanced. Thus glial-neuronal interactions, specifically during pruning are important for the patterning of adult innervation. Our studies also suggest that FasII plays a role in mediating this communication. At the end of the pruning phase, FasII localizes to glia, which envelops each of the stabilized contact points. When glial FasII levels are increased using the Gal4/UAS system of targeted expression, pruning of secondary branches is enhanced. Our results indicate that glia regulate pruning of secondary branches by influencing the balance between stabilization and pruning. This was confirmed by an observed rescue of the innervation phenotype of FasII hypomorphs by over expressing FasII in glia.     

Prospero maintains the mitotic potential of glial precursors enabling them to respond to neurons

During central nervous system development, glial cells need to be in the correct number and location, at the correct time, to enable axon guidance and neuropile formation. Repair of the injured or diseased central nervous system will require the manipulation of glial precursors, so that the number of glial cells is adjusted to that of neurons, enabling axonal tracts to be rebuilt, remyelinated and functional. Unfortunately, the molecular mechanisms controlling glial precursor proliferative potential are unknown. We show here that glial proliferation is regulated by interactions with axons and that the Drosophila gene prospero is required to maintain the mitotic potential of glia. During growth cone guidance, Prospero positively regulates cycE promoting cell proliferation. Neuronal Vein activates the MAPKinase signalling pathway in the glia with highest Prospero levels, coupling axon extension with glial proliferation. Later on, Prospero maintains glial precursors in an undifferentiated state by activating Notch and antagonising the p27/p21 homologue Dacapo. This enables prospero-expressing cells alone to divide further upon elimination of neurons and to adjust glial number to axons during development.     

Development of the Drosophila mushroom bodies: sequential generation of three distinct types of neurons from a neuroblast.

The mushroom bodies (MBs) are prominent structures in the Drosophila brain that are essential for olfactory learning and memory. Characterization of the development and projection patterns of individual MB neurons will be important for elucidating their functions. Using mosaic analysis with a repressible cell marker (Lee, T. and Luo, L. (1999) Neuron 22, 451-461), we have positively marked the axons and dendrites of multicellular and single-cell mushroom body clones at specific developmental stages. Systematic clonal analysis demonstrates that a single mushroom body neuroblast sequentially generates at least three types of morphologically distinct neurons. Neurons projecting into the (gamma) lobe of the adult MB are born first, prior to the mid-3rd instar larval stage. Neurons projecting into the alpha' and beta' lobes are born between the mid-3rd instar larval stage and puparium formation. Finally, neurons projecting into the alpha and beta lobes are born after puparium formation. Visualization of individual MB neurons has also revealed how different neurons acquire their characteristic axon projections. During the larval stage, axons of all MB neurons bifurcate into both the dorsal and medial lobes. Shortly after puparium formation, larval MB neurons are selectively pruned according to birthdays. Degeneration of axon branches makes early-born gamma neurons retain only their main processes in the peduncle, which then project into the adult gamma lobe without bifurcation. In contrast, the basic axon projections of the later-born (alpha'/beta') larval neurons are preserved during metamorphosis. This study illustrates the cellular organization of mushroom bodies and the development of different MB neurons at the single cell level. It allows for future studies on the molecular mechanisms of mushroom body development.     

An axon scaffold induced by retinal axons directs glia to destinations in the Drosophila optic lobe

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. Glial migration conversely depends on photoreceptor axons, as glia precursors stall in their progenitor zones when retinal innervation is eliminated. Our 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 towards 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.     

Roundabout signalling, cell contact and trophic support confine longitudinal glia and axons in the Drosophila CNS

Contrary to our knowledge of the genetic control of midline crossing, the mechanisms that generate and maintain the longitudinal axon pathways of the Drosophila CNS are largely unknown. The longitudinal pathways are formed by ipsilateral pioneer axons and the longitudinal glia. The longitudinal glia dictate these axonal trajectories and provide trophic support to later projecting follower neurons. Follower interneuron axons cross the midline once and join these pathways to form the longitudinal connectives. Once on the contralateral side, longitudinal axons are repelled from recrossing the midline by the midline repulsive signal Slit and its axonal receptor Roundabout. We show that longitudinal glia also transiently express roundabout, which halts their ventral migration short of the midline. Once in contact with axons, glia cease to express roundabout and become dependent on neurons for their survival. Trophic support and cell-cell contact restrict glial movement and axonal trajectories. The significance of this relationship is revealed when neuron-glia interactions are disrupted by neuronal ablation or mutation in the glial cells missing gene, which eliminates glia, when axons and glia cross the midline despite continued midline repellent signalling.     

Ex vivo Culturing of Whole,Developing Drosophila Brains

We describe a method for ex vivo culturing of whole Drosophila brains. This can be used as a counterpoint to chronic genetic manipulations for investigating the cell biology and development of central brain structures by allowing acute pharmacological interventions and live imaging of cellular processes. As an example of the technique, prior work from our lab¹ has shown that a previously unrecognized subcellular compartment lies between the axonal and somatodendritic compartments of axons of the Drosophila central brain. The development of this compartment, referred to as the axon initial segment (AIS)², was shown genetically to depend on the neuron-specific cyclin-dependent kinase, Cdk5. We show here that ex vivo treatment of wild-type Drosophila larval brains with the Cdk5-specific pharmacological inhibitors roscovitine and olomoucine³ causes acute changes in actin organization, and in localization of the cell-surface protein Fasciclin 2, that mimic the changes seen in mutants that lack Cdk5 activity genetically.A second example of the ex vivo culture technique is provided for remodeling of the connections of embryonic mushroom body (MB) gamma neurons during metamorphosis from larva to adult. The mushroom body is the center of olfactory learning and memory in the fly⁴, and these gamma neurons prune their axonal and dendritic branches during pupal development and then re-extend branches at a later timepoint to establish the adult innervation pattern⁵. Pruning of these neurons of the MB has been shown to occur via local degeneration of neurite branches⁶, by a mechanism that is triggered by ecdysone, a steroid hormone, acting at the ecdysone receptor B1⁷, and that is dependent on the activity of the ubiquitin-proteasome system⁶. Our method of ex vivo culturing can be used to interrogate further the mechanism of developmental remodeling. We found that in the ex vivo culture setting, gamma neurons of the MB recapitulated the process of developmental pruning with a time course similar to that in vivo. It was essential, however, to wait until 1.5 hours after puparium formation before explanting the tissue in order for the cells to commit irreversibly to metamorphosis; dissection of animals at the onset of pupariation led to little or no metamorphosis in culture. Thus, with appropriate modification, the ex vivo culture approach can be applied to study dynamic as well as steady state aspects of central brain biology.     

DPP signaling controls development of the lamina glia required for retinal axon targeting in the visual system of Drosophila

The Drosophila visual system consists of the compound eyes and the optic ganglia in the brain. Among the eight photoreceptor (R) neurons, axons from the R1-R6 neurons stop between two layers of glial cells in the lamina, the most superficial ganglion in the optic lobe. Although it has been suggested that the lamina glia serve as intermediate targets of R axons, little is known about the mechanisms by which these cells develop. We show that DPP signaling plays a key role in this process. dpp is expressed at the margin of the lamina target region, where glial precursors reside. The generation of clones mutant for Medea, the DPP signal transducer, or inhibition of DPP signaling in this region resulted in defects in R neuron projection patterns and in the lamina morphology, which was caused by defects in the differentiation of the lamina glial cells. glial cells missing/glial cells deficient (gcm; also known as glide) is expressed shortly after glia precursors start to differentiate and migrate. Its expression depends on DPP; gcm is reduced or absent in dpp mutants or Medea clones, and ectopic activation of DPP signaling induces ectopic expression of gcm and REPO. In addition, R axon projections and lamina glia development were impaired by the expression of a dominant-negative form of gcm, suggesting that gcm indeed controls the differentiation of lamina glial cells. These results suggest that DPP signaling mediates the maturation of the lamina glia required for the correct R axon projection pattern by controlling the expression of gcm.     

Wallerian degeneration of zebrafish trigeminal axons in the skin is required for regeneration and developmental pruning

Fragments of injured axons that detach from their cell body break down by the molecularly regulated process of Wallerian degeneration (WD). Although WD resembles local axon degeneration, a common mechanism for refining neuronal structure, several previously examined instances of developmental pruning were unaffected by WD pathways. We used laser axotomy and time-lapse confocal imaging to characterize and compare peripheral sensory axon WD and developmental pruning in live zebrafish larvae. Detached fragments of single injured axon arbors underwent three stereotyped phases of WD: a lag phase, a fragmentation phase and clearance. The lag phase was developmentally regulated, becoming shorter as embryos aged, while the length of the clearance phase increased with the amount of axon debris. Both cell-specific inhibition of ubiquitylation and overexpression of the Wallerian degeneration slow protein (Wld(S)) lengthened the lag phase dramatically, but neither affected fragmentation. Persistent Wld(S)-expressing axon fragments directly repelled regenerating axon branches of their parent arbor, similar to self-repulsion among sister branches of intact arbors. Expression of Wld(S) also disrupted naturally occurring local axon pruning and axon degeneration in spontaneously dying trigeminal neurons: although pieces of Wld(S)-expressing axons were pruned, and some Wld(S)-expressing cells still died during development, in both cases detached axon fragments failed to degenerate. We propose that spontaneously pruned fragments of peripheral sensory axons must be removed by a WD-like mechanism to permit efficient innervation of the epidermis.     

Glia dictate pioneer axon trajectories in the Drosophila embryonic CNS

Whereas considerable progress has been made in understanding the molecular mechanisms of axon guidance across the midline, it is still unclear how the axonal trajectories of longitudinal pioneer neurons, which never cross the midline, are established. Here we show that longitudinal glia of the embryonic Drosophila CNS direct formation of pioneer axon pathways. By ablation and analysis of glial cells missing mutants, we demonstrate that glia are required for two kinds of processes. Firstly, glia are required for growth cone guidance, although this requirement is not absolute. We show that the route of extending growth cones is rich in neuronal cell bodies and glia, and also in long processes from both these cell types. Interactions between neurons, glia and their long processes orient extending growth cones. Secondly, glia direct the fasciculation and defasciculation of axons, which pattern the pioneer pathways. Together these events are essential for the selective fasciculation of follower axons along the longitudinal pathways.     

Generation and early differentiation of glial cells in the first optic ganglion of Drosophila melanogaster.

We have examined the generation and development of glial cells in the first optic ganglion, the lamina, of Drosophila melanogaster. Previous work has shown that the growth of retinal axons into the developing optic lobes induces the terminal cell divisions that generate the lamina monopolar neurons. We investigated whether photoreceptor ingrowth also influences the development of lamina glial cells, using P element enhancer trap lines, genetic mosaics and birthdating analysis. Enhancer trap lines that mark the differentiating lamina glial cells were found to require retinal innervation for expression. In mutants with only a few photoreceptors, only the few glial cells near ingrowing axons expressed the marker. Genetic mosaic analysis indicates that the lamina neurons and glial cells are readily separable, suggesting that these are derived from distinct lineages. Additionally, BrdU pulse-chase experiments showed that the cell divisions that produce lamina glia, unlike those producing lamina neurons, are not spatially or temporally correlated with the retinal axon ingrowth. Finally, in mutants lacking photoreceptors, cell divisions in the glial lineage appeared normal. We conclude that the lamina glial cells derive from a lineage that is distinct from that of the L-neurons, that glia are generated independently of photoreceptor input, and that completion of the terminal glial differentiation program depends, directly or indirectly, on an inductive signal from photoreceptor axons.     

The Drosophila neuregulin vein maintains glial survival during axon guidance in the CNS.

Neuron-glia interactions are necessary for the formation of the longitudinal axon trajectories in the Drosophila central nervous system. Longitudinal glial cells are required for axon guidance and fasciculation, and pioneer neurons for trophic support of the glia. Neuregulin is a neuronal molecule that controls glial survival in the vertebrate nervous system. The Drosophila protein Vein has structural similarities with Neuregulin. We show here that Vein functions like a Neuregulin to maintain glial cell survival. We present direct in vivo evidence at single-cell resolution that Vein is produced by pioneer neurons and maintains the survival of neighboring longitudinal glia. This mechanism links axon guidance to control of glial cell number and may contribute to plasticity during the establishment of normal axonal trajectories.