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.     

Glia maintain follower neuron survival during Drosophila CNS development

While survival of CNS neurons appears to depend on multiple neuronal and non-neuronal factors, it remains largely unknown how neuronal survival is controlled during development. Here we show that glia regulate neuronal survival during formation of the Drosophila embryonic CNS. When glial function is impaired either by mutation of the glial cells missing gene, which transforms glia toward a neuronal fate, or by targeted genetic glial ablation, neuronal death is induced non-autonomously. Pioneer neurons, which establish the first longitudinal axon fascicles, are insensitive to glial depletion whereas the later extending follower neurons die. This differential requirement of neurons for glia is instructive in patterning and links control of cell number with axon guidance during CNS development.     

Glial cell regulation of neurotransmission and behavior in Drosophila

Mounting evidence demonstrates that glial cells might have important roles in regulating the physiology and behavior of adult animals. We summarize some of this evidence here, with an emphasis on the roles of glia of the differentiated nervous system in controlling neuronal excitability, behavior and plasticity. In the review we highlight studies in Drosophila and discuss results from the analysis of mammalian astrocytes that demonstrate roles for glia in the adult nervous system.     

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.     

Characterization of two novel lipocalins expressed in the Drosophila embryonic nervous system

We have found two novel lipocalins in the fruit fly Drosophila melanogaster that are homologous to the grasshopper Lazarillo, a singular lipocalin within this protein family which functions in axon guidance during nervous system development. Sequence analysis suggests that the two Drosophila proteins are secreted and possess peptide regions unique in the lipocalin family. The mRNAs of DNLaz (for Drosophila neural Lazarillo) and DGLaz (for Drosophila glial Lazarillo) are expressed with different temporal patterns during embryogenesis. They show low levels of larval expression and are highly expressed in pupa and adult flies. DNLaz mRNA is transcribed in a subset of neurons and neuronal precursors in the embryonic CNS. DGLaz mRNA is found in a subset of glial cells of the CNS: the longitudinal glia and the medial cell body glia. Both lipocalins are also expressed outside the nervous system in the developing gut, fat body and amnioserosa. The DNLaz protein is detected in a subset of axons in the developing CNS. Treatment with a secretion blocker enhances the antibody labeling, indicating the DNLaz secreted nature. These findings make the embryonic nervous system expression of lipocalins a feature more widespread than previously thought. We propose that DNLaz and DGLaz may have a role in axonal outgrowth and pathfinding, although other putative functions are also discussed.     

Spatially discrete FGF-mediated signalling directs glial morphogenesis.

Neurons provide critical signals that regulate both the number and differentiation of glia. In addition, glia are attracted to and enwrap neuronal axonal processes. FGF-like signalling is thought to be one of the many potential axon-derived morphogenetic signals, however, the multiple roles of FGFs have made experimental tests of these signals difficult in vivo. In the Drosophila FGF receptor mutant heartless, glia migrate to axons, but fail to elongate around them. This study shows that in the similar but larger grasshopper CNS, FGF signalling is likely to mediate one step in the close interaction between glia and axons. FGF2-coated beads attract glia in the CNS and compete with axons for their resident, enwrapped glia. In addition, bath applied FGF2 causes mature axonal glia, which normally enwrap axon tracts, to round up. FGF2 activates the product of the grasshopper heartless FGF receptor gene and probably interferes with the normal function of an endogenous axon-associated FGF-like molecule. It is proposed that insect axons provide a critical spatially restricted FGF-like signal that induces glia to enwrap them.     

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.     

Neuron-glia interaction in the insect nervous system

In all complex organisms, glial cells are pivotal for neuronal development and function. Insects are characterized by having only a small number of these cells, which nevertheless display a remarkable molecular diversity. An intricate relationship between neurons and glia is initially required for glial migration and during axonal patterning. Recent data suggest that in organisms such as Drosophila, a prime role of glial cells lies in setting boundaries to guide and constrain axonal growth.     

Morphogenesis and proliferation of the larval brain glia in Drosophila

Glial cells subserve a number of essential functions during development and function of the Drosophila brain, including the control of neuroblast proliferation, neuronal positioning and axonal pathfinding. Three major classes of glial cells have been identified. Surface glia surround the brain externally. Neuropile glia ensheath the neuropile and form septa within the neuropile that define distinct neuropile compartments. Cortex glia form a scaffold around neuronal cell bodies in the cortex. In this paper we have used global glial markers and GFP-labeled clones to describe the morphology, development and proliferation pattern of the three types of glial cells in the larval brain. We show that both surface glia and cortex glia contribute to the glial layer surrounding the brain. Cortex glia also form a significant part of the glial layer surrounding the neuropile. Glial cell numbers increase slowly during the first half of larval development but show a rapid incline in the third larval instar. This increase results from mitosis of differentiated glia, but, more significantly, from the proliferation of neuroblasts.     

The scoop on the fly brain: glial engulfment functions in Drosophila

Glial cells provide support and protection for neurons in the embryonic and adult brain, mediated in part through the phagocytic activity of glia. Glial cells engulf apoptotic cells and pruned neurites from the developing nervous system, and also clear degenerating neuronal debris from the adult brain after neural trauma. Studies indicate that Drosophila melanogaster is an ideal model system to elucidate the mechanisms of engulfment by glia. The recent studies reviewed here show that many features of glial engulfment are conserved across species and argue that work in Drosophila will provide valuable cellular and molecular insight into glial engulfment activity in mammals.     

The effect of extrinsic Wnt/β‐catenin signaling in Muller glia on retinal ganglion cell neurite growth

Muller glia are the predominant glial cell type in the retina, and they structurally and metabolically support retinal neurons. Wnt/β‐catenin signaling pathways play essential roles in the central nervous system, including glial and neuronal differentiation, axonal growth, and neuronal regeneration. We previously demonstrated that Wnt signaling activation in retinal ganglion cells (RGC) induces axonal regeneration after injury. However, whether Wnt signaling within the adjacent Muller glia plays an axongenic role is not known. In this study, we characterized the effect of Wnt signaling in Muller glia on RGC neurite growth. Primary Muller glia and RGC cells were grown in transwell co‐cultures and adenoviral constructs driving Wnt regulatory genes were used to activate and inhibit Wnt signaling specifically in primary Muller glia. Our results demonstrated that activation of Wnt signaling in Muller glia significantly increased RGC average neurite length and branch site number. In addition, the secretome of Muller glia after induction or inhibition of Wnt signaling was characterized using protein profiling of conditioned media by Q Exactive mass spectrometry. The Muller glia secretome after activation of Wnt signaling had distinct and more numerous proteins involved in regulation of axon extension, axon projection and cell adhesion. Furthermore, we showed highly redundant expression of Wnt signaling ligands in Muller glia and Frizzled receptors in RGCs and Muller glia. Therefore, this study provides new information about potential neurite growth promoting molecules in the Muller glia secretome, and identified Wnt‐dependent target proteins that may mediate the axonal growth.     

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.     

Extracellular matrix: functions in the nervous system

An astonishing number of extracellular matrix glycoproteins are expressed in dynamic patterns in the developing and adult nervous system. Neural stem cells, neurons, and glia express receptors that mediate interactions with specific extracellular matrix molecules. Functional studies in vitro and genetic studies in mice have provided evidence that the extracellular matrix affects virtually all aspects of nervous system development and function. Here we will summarize recent findings that have shed light on the specific functions of defined extracellular matrix molecules on such diverse processes as neural stem cell differentiation, neuronal migration, the formation of axonal tracts, and the maturation and function of synapses in the peripheral and central nervous system.     

Tissue plasminogen activator-mediated fibrinolysis protects against axonal degeneration and demyelination after sciatic nerve injury

Tissue plasminogen activator (tPA) is a serine protease that converts plasminogen to plasmin and can trigger the degradation of extracellular matrix proteins. In the nervous system, under noninflammatory conditions, tPA contributes to excitotoxic neuronal death, probably through degradation of laminin. To evaluate the contribution of extracellular proteolysis in inflammatory neuronal degeneration, we performed sciatic nerve injury in mice. Proteolytic activity was increased in the nerve after injury, and this activity was primarily because of Schwann cell-produced tPA. To identify whether tPA release after nerve damage played a beneficial or deleterious role, we crushed the sciatic nerve of mice deficient for tPA. Axonal demyelination was exacerbated in the absence of tPA or plasminogen, indicating that tPA has a protective role in nerve injury, and that this protective effect is due to its proteolytic action on plasminogen. Axonal damage was correlated with increased fibrin(ogen) deposition, suggesting that this protein might play a role in neuronal injury. Consistent with this idea, the increased axonal degeneration phenotype in tPA- or plasminogen-deficient mice was ameliorated by genetic or pharmacological depletion of fibrinogen, identifying fibrin as the plasmin substrate in the nervous system under inflammatory axonal damage. This study shows that fibrin deposition exacerbates axonal injury, and that induction of an extracellular proteolytic cascade is a beneficial response of the tissue to remove fibrin. tPA/plasmin-mediated fibrinolysis may be a widespread protective mechanism in neuroinflammatory pathologies.     

Post-natal knockout of prion protein alters hippocampal CA1 properties, but does not result in neurodegeneration

Prion protein (PrP) plays a crucial role in prion disease, but its physiological function remains unclear. Mice with gene deletions restricted to the coding region of PrP have only minor phenotypic deficits, but are resistant to prion disease. We generated double transgenic mice using the Cre-loxP system to examine the effects of PrP depletion on neuronal survival and function in adult brain. Cre-mediated ablation of PrP in neurons occurred after 9 weeks. We found that the mice remained healthy without evidence of neurodegeneration or other histopathological changes for up to 15 months post-knockout. However, on neurophysiological evaluation, they showed significant reduction of afterhyperpolarization potentials (AHPs) in hippocampal CA1 cells, suggesting a direct role for PrP in the modulation of neuronal excitability. These data provide new insights into PrP function. Furthermore, they show that acute depletion of PrP does not affect neuronal survival in this model, ruling out loss of PrP function as a pathogenic mechanism in prion disease and validating therapeutic approaches targeting PrP.     

Peripheral glia direct axon guidance across the CNS/PNS transition zone.

CNS glia have integral roles in directing axon migration of both vertebrates and insects. In contrast, very little is known about the roles of PNS glia in axonal pathfinding. In vertebrates and Drosophila, anatomical evidence shows that peripheral glia prefigure the transition zones through which axons migrate into and out of the CNS. Therefore, peripheral glia could guide axons at the transition zone. We used the Drosophila model system to test this hypothesis by ablating peripheral glia early in embryonic neurodevelopment via targeted overexpression of cell death genes grim and ced-3. The effects of peripheral glial loss on sensory and motor neuron development were analyzed. Motor axons initially exit the CNS in abnormal patterns in the absence of peripheral glia. However, they must use other cues within the periphery to find their correct target muscles since early pathfinding errors are largely overcome. When peripheral glia are lost, sensory axons show disrupted migration as they travel centrally. This is not a result of motor neuron defects, as determined by motor/sensory double-labeling experiments. We conclude that peripheral glia prefigure the CNS/PNS transition zone and guide axons as they traverse this region.     

Two distinct mechanisms segregate Prospero in the longitudinal glia underlying the timing of interactions with axons

Prospero is required in dividing longitudinal glia (LG) during axon guidance; initially to enable glial division in response to neuronal contact, and subsequently to maintain glial precursors in a quiescent state with mitotic potential. Only Prospero-positive LG respond to neuronal ablation by over-proliferating, mimicking a glial-repair response. Prospero is distributed unequally through the progeny cells of the longitudinal glioblast lineage. Just before axon contact the concentration of Prospero is higher in two of the four progeny cells, and after axon guidance Prospero is present only in six out of ten progeny LG. Here we ask how Prospero is distributed unequally in these two distinct phases. We show that before neuronal contact, longitudinal glioblasts undergo invaginating divisions, perpendicular to the ectodermal layer. Miranda is required to segregate Prospero asymmetrically up to the four glial-progeny stage. After neuronal contact, Prospero is present in only the LG that activate Notch signalling in response to Serrate provided by commissural axons, and Numb is restricted to the glia that do not contain Prospero. As a result of this dual regulation of Prospero deployment, glia are coupled to the formation and maintenance of axonal trajectories.     

A Global In Vivo Drosophila RNAi Screen Identifies a Key Role of Ceramide Phosphoethanolamine for Glial Ensheathment of Axons

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

Misguided axonal projections,neural cell adhesion molecule 180 mRNA upregulation,and altered behavior in mice deficient for the close homolog of L1

Cell recognition molecules are involved in nervous system development and participate in synaptic plasticity in the adult brain. The close homolog of L1 (CHL1), a recently identified member of the L1 family of cell adhesion molecules, is expressed by neurons and glia in the central nervous system and by Schwann cells in the peripheral nervous system in a pattern overlapping, but distinct from, the other members of the L1 family. In humans, CHL1 (also referred to as CALL) is a candidate gene for 3p- syndrome-associated mental impairment. In the present study, we generated and analyzed CHL1-deficient mice. At the morphological level, these mice showed alterations of hippocampal mossy fiber organization and of olfactory axon projections. Expression of the mRNA of the synapse-specific neural cell adhesion molecule 180 isoform was upregulated in adult CHL1-deficient mice, but the mRNA levels of several other recognition molecules were not changed. The behavior of CHL1-deficient mice in the open field, the elevated plus maze, and the Morris water maze indicated that the mutant animals reacted differently to their environment. Our data show that the permanent absence of CHL1 results in misguided axonal projections and aberrant axonal connectivity and alters the exploratory behavior in novel environments, suggesting deficits in information processing in CHL1-deficient mice.