The effects of an anti-mitotic drug,bleomycin, on glial repair in an insect central nervous system

Summary The DNA-binding drug, bleomycin, has a profound effect on neural repair following selective glial disruption by ethidium bromide. The contribution of the granule-containing cells (which normally appear in the early stages of repair) is greatly reduced, the restoration of the blood-brain barrier is delayed and the ultrastructural organization of the reorganising perineurium is dramatically changed. The aberrant perineurial structure and function observed in the presence of bleomycin are postulated to result from the effects of the drug on haemocytes which, together with endogenous reactive cells, contribute to the normal process of glial repair.     

Glial repair in the cultured central nervous system of an insect

Summary Insect glial cells are capable of division and repair in organ culture after selective damage with the toxin ethidium bromide. The repair is slower and less organised than seen in vivo after similar treatment and is still incomplete after one month. Granule-containing cells, which play an important role in the early stages of repair in vivo, are never seen in cultured connectives. This observation adds further support to the hypothesis that these cells are derived from haemocytes and that their presence is necessary for rapid and orderly repair. The uptake of ³H-thymidine into perineurial glial cells in vitro, both in control and ethidiumtreated connectives, shows that there is a considerable proliferation of cells in this region. Some uptake of thymidine is also seen in subperineurial glia but division alone cannot account for the large increase in the number of glial nuclei found at the early stages of repair in this region. Further, glial cells with diverse morphologies suggest that subpopulations are present. We conclude that cell migration from undamaged areas, as well as cell proliferation, is necessary for CNS repair in vitro.     

Blood cells contribute to glial repair in an insect

Using antibodies specific for haemocytes, we have shown that these blood cells penetrate the abdominal nervous connectives of the cockroach following selective disruption of the glia using the DNA-intercalating drug, ethidium bromide, as a glial toxin. Within 4 days post-lesion, the labelled cells formed a mosaic beneath the neural lamella and penetrated deeply among the disrupted subperineurial glia. These observations confirm that exogenous cells are involved in glial repair and support a previous hypothesis that they play critical roles in both structural repair and the recruitment of endogenous reactive cells.     

Glial repair in an insect

The repair of cockroach central nervous connectives, following selective glial disruption, involves an initial invasion of the lesion by a novel cell class. The available evidence, including that obtained using monoclonal antibodies, shows that these cells arise from circulating haemocytes. These invasive exogenous cells are restricted to the lesion zone. They are not only involved in initial repair of the peripheral glial elements, but may also be responsible for initiating recruitment and division of endogenous reactive cells. There is a clear anterior polarity in this recruitment, with significantly higher numbers of cells appearing anterior to, and then within, the lesion area. Characteristically, recognizable exogenous cells decline in number after 3 days, although there is no overall reduction in cell numbers within the lesion at this stage, nor has significant cell division begun. This suggests that the haemocyte-derived cells transform into, or are replaced by, functional perineurial glia, between 3 and 5 days, coincident with the restoration of the blood-brain barrier and the onset of endogenous cell division. Glial repair in the insect CNS can thus be divided into three phases which show striking similarities to the repair sequence in vertebrate brain. These include: an initial invasion of the lesion by exogenous cells, subsequent glial proliferation and then longer term fluxes in cell numbers and distribution.     

Detection of glial fibrillary acidic protein (GFAP) and vimentin (Vim) by immunoelectron microscopy of the glial cells in the central nervous system of the snail Megalobulimus abbreviatus

When examined under an electron microscope, the central nervous system of Megalobulimus abbreviatus showed two types of glial cells: firstly, protoplasmic glial cells which displayed a nucleus with peripheral heterochromatin, scanty or no intermediate filaments, a developed Golgi complex, rough and smooth endoplasmic reticula, mitochondria and polymorphic lysosomes that indicate phagocytic activity of debris from the extracellular space; and, secondly, fibrous glial cells which showed numerous glial fibrillary acidic protein (GFAP) and vimentin immunoreactive intermediate filament bundles, a discrete Golgi complex, mitochondria, endoplasmic reticulum, lipid droplets and lysosomes. The contacts between the glial cells consisted of desmosomes and puncta adherentia, while those between the glial cells and the basal lamina consisted of hemidesmosomes. Both glial cell types were located in the cortex and medullary regions, however, the protoplasmic glial cells prevailed in the cortical region, while the fibrous glial cells prevailed in the medullar region. As the nervous tissue is avascular, the passage of nutrients and waste products may be facilitated by the glial labyrinthic system which is located in the cortical region. Glial processes adjacent to large and giant neurones formed a trophospongium, which seemed to be involved in a metabolic exchange between these cells. Thus, this evidence suggests that glial cells of M. abbreviatus are involved in structural support, isolation of different ganglionic areas, the formation of a microcirculatory system and an intimate metabolic relationship with neurones.     

Neural repair in an insect: cell recruitment and deployment following selective glial disruption

Summary In Periplaneta americana, SEM of abdominal nervous connectives revealed a rapid accumulation of haemocytes on the surface of the neural lamella within 24 h of selective disruption of the underlying neuroglia by ethidium bromide. After 4 days the neural lamella was effectively clear of adhering haemocytes, but showed characteristic blisters, which, it is postulated, represented the points of entry of the cells from the haemocoel into the underlying tissues. A notable subsequent feature was a substantial increase in the number of cells within repairing connectives. Initially, there was a marked asymmetry in their distribution, with significantly higher numbers of cells anterior to, and within, the lesion area. It seems likely that this polarity resulted from differential cell division within the connectives. The initial asymmetry disappeared after seven days. However, increased perineurial cell numbers were maintained in the lesion area after one month and were still apparent two months after selective glial disruption. There was no equivalent increase in cell numbers in the lesion zone of cultured cords or, in vivo, after injection of the DNA-scission drug, bleomycin, treatments which preclude haemocyte involvement. It is suggested that in the absence of haemocytes and with suppression of proliferation by endogenous cells, repair is achieved by redeployment or growth of adjacent, undamaged glia.     

Adult insect glial culture: Activation, substrate effects and proliferation

Glial cells from an adult insect, Periplaneta americana, have been grown in neurone-free cultures. No growth occurred from freshly-excised fragments of abdominal nervous connectives. Vigorous growth was obtained, however, from explants of connectives induced to proliferate by prior exposure to a toxin, ethidium bromide, applied selectively to glial cells in vivo. Glial growth in vitro is dependent upon the initiation of early stages of repair in vivo: this supports the idea that haemocytes which invade the lesion zone immediately after damage are involved in directing proliferation of perineurial and sub-perineurial glia. In contrast, both glial and neuronal cells grew in vitro from explanted abdominal ganglia without prior glial lesioning, indicating that different factors may determine cellular regeneration in this domain. The morphology of the proliferating cells was influenced by the substrate; extensive glial migration was restricted to areas of close contact between cell and substrate surface.     

Olfactory ensheathing cells: their role in central nervous system repair

The olfactory system is an unusual tissue in that it can support neurogenesis throughout life; permitting the in-growth and synapse formation of olfactory receptor axons into the central nervous system (CNS) environment of the olfactory bulb. It is thought that this unusual property is in part due to the olfactory glial cells, termed olfactory ensheathing cells (OECs), but also due to neuronal stem cells. These glial cells originate from the olfactory placode and possess many properties in common with the glial cells from the peripheral nervous system (PNS), Schwann cells. Recent data has suggested that olfactory ensheathing cells are a distinct glial cell type and possess properties, which might make them more suitable for transplant-mediated repair of central nervous system injury models. This paper reviews the biological properties of these cells and illustrates their use in central nervous system repair.     

Cell recruitment during glial repair: the role of exogenous cells

Summary Selective disruption of the neuroglia in penultimate abdominal connectives of the cockroach nerve is followed by a rapid accumulation of cells in the perineurial layer of the lesion. Subsequently, there is an abrupt, secondary, rise in cell numbers in the undamaged perineurial tissues, anterior to the lesion and adjacent to the 4th abdominal ganglia. By 7 days the increased cell numbers are again effectively confined to the original lesion zone. The initial rise in cell numbers is postulated to result from an invasion by blood-borne haemocytes and the subsequent increase, in undamaged perineurial tissues, from the mobilization of endogenous reactive cells. Recruitment of the endogenous cells is inhibited if the haemocytes are excluded from the lesion. There is a slower mobilization of sub-perineurial cells, which, again, is inhibited following exclusion of haemocytes from the lesion zone. It is postulated that the recruitment of the endogenous reactive cells is initiated by the invading haemocytes which transform to granule-containing cells and release diffusible morphogenic and/or mitogenic factors.     

BrdU incorporation reveals DNA replication in non dividing glial cells in the larval abdominal CNS ofDrosophila

Glial cells are of significant importance for central nervous system development and function. In insects, knowledge of the types and development of CNS glia is rather low. This is especially true for postembryonic glial development. Using bromodeoxyuridine incorporation and enhancer trap lines we identified a reproducible spatial and temporal pattern of DNA replicating cells in the abdominal larval CNS (A3-7 neuromeres) ofDrosophila melanogaster. These cells correspond to embryonically established glial cells in that region. Except for a specific subfraction, these cells apparently do not divide during larval life. Similar patterns were found in two otherDrosophila species,D. virilis andD. hydei.     

Drosophila glial development is regulated by genes involved in the control of neuronal cell fate

The Drosophila proneural genes specify neuronal determination among cells within the ectoderm. Here we address the question of whether proneural genes also affect the specification of glia, the most abundant cell type in the nervous system. We provide evidence that the proneural gene daughterless is essential for the formation of two major classes of PNS glia. In contrast, the proneural genes in the achaete-scute complex have no detectable effect on the specification and differentiation of these PNS glia and certain CNS glia. We also show that, as with neuronal development, glial determination is restricted by the neurogenic genes neuralized, Delta, and the genes of the Enhancer of split complex. Finally, we demonstrate that prospero, a gene involved in neuronal differentiation, also affects glial development. These results demonstrate extensive overlap in the genetic control of glial and neuronal development.Abbreviations ß galactosidase - (ß-gal) Alkaline phosphatase - (AP) Central nervous system - (CNS) Peripheral nervous system - (PNS) Home domain binding sites - (HDS) Helix-loop-helix - (HLH) Peripheral glia - (PG) Exit glia - (EG) Dorsal roof glia - (DRG) Intersegmental glia - (ISG) Midline glia - (MG) chordotonal - (CH) Sensory mother cell     

Ultrastructural localization of lectin-binding sites and laminin-like immunoreactivity in glial cells and neurites growing out from explant cultures of the central nervous system of embryonic locusts

Summary Lectins with different sugar specificities and labeled with horseradish peroxidase or gold were used to study, at the electron-microscopic level, surface glycoconjugates of glial cells and neurites growing out from explant cultures of the central nervous system of embryonic locusts. Differential binding to differentiating glial cells and to neurites was demonstrated. Concanavalin A (Con A) and wheat-germ agglutinin (WGA) bound to glial and neurite surfaces with different degrees of labeling. The formation of glial processes and junctional complexes was invariably accompanied by a corresponding increase of Con A- and WGA-receptors. Peanut agglutinin (PNA) failed to bind to glial cells but strongly stained the plasma membrane of neurite junctions. Lotus tetragonolobus a. (LTA) did not bind either to glial cells or to neurites. In addition, staining with an antibody against laminin showed labeling in areas of neurite outgrowth and neurite interactions; this resembled the localization of PNA receptors. These findings provide evidence for the presence of different carbohydrates at the surface of neurites and glial cells of locust. Their predominant localization in glial processes and neurite junctions suggests that these carbohydrates constitute part of a group adhesion glycoproteins that also includes laminin.     

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

The neural stem cells that give rise to the neural lineages of the brain can generate their progeny directly or through transit amplifying intermediate neural progenitor cells (INPs). The INP-producing neural stem cells in Drosophila are called type II neuroblasts, and their neural progeny innervate the central complex, a prominent integrative brain center. Here we use 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. Our 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.     

Embryonic expression of Drosophila ceramide synthase schlank in developing gut, CNS and PNS

Schlank is a member of the highly conserved ceramide synthase family and controls growth and body fat in Drosophila. Ceramide synthases are key enzymes in the sphingolipid de novo synthesis pathway. Ceramide synthase proteins and the (dihydro)ceramide produced are involved in a variety of biological processes among them apoptosis and neurodegeneration. The full extent of their involvement in these processes will require a precise analysis of the distribution and expression pattern of ceramide synthases. Paralogs of the ceramide synthase family have been found in all eukaryotes studied, however the mRNA and protein expression patterns have not yet been analysed systematically. In this study, we use antibodies that specifically recognize Schlank, a schlank mRNA probe and an endogenous schlank promoter driven LacZ reporter line to reveal the expression pattern of Schlank throughout embryogenesis. We found that Schlank is expressed in all embryonic epithelia during embryogenesis including the developing epidermis and the gastrointestinal tract. In addition, Schlank is upregulated in the developing central (CNS) and peripheral nervous system (PNS). Co-staining experiments with neuronal and glial markers revealed specific expression of Schlank in glial and neuronal cells of the CNS and PNS.     

Dye-filling of the amphid sheath glia: implications for the functional relationship between sensory neurons and glia in Caenorhabditis elegans

The nervous system is composed of cells including neurons and glia. It has been believed that the former cells play central roles in various neural functions while the latter ones have only supportive functions for neurons. However, recent findings suggest that glial cells actively participate in neural activities, and the cooperation between neurons and glia is important for nervous system functions. In Caenorhabditis elegans, amphid sensory organs in the head also consist of sensory neurons and glia-like support cells (amphid socket and amphid sheath cells). Ciliary endings of some sensory neurons exposed to the environment detect various chemicals, molecules and signals, and the cilia of some neurons can also take up fluorescent dyes such as DiI. Here, we show that the amphid sheath glia are also stained with DiI and that its uptake by the amphid sheath cells correlates with DiI-filling of sensory neurons, suggesting that the amphid sheath glia might interact with sensory neurons. Furthermore, the localization of the amphid sheath cell reporter F52E1.2SP::YFP is abnormal in che-2 mutants, which have defective cilia. These findings imply that sensory neurons might affect amphid sheath glia functions in the amphid sensory organ of C. elegans.     

Two lectins on the surface of Helix pomatia haemocytes: a Ca2+-dependent,GalNac-specific lectin and a Ca2+-independent,mannose 6-phosphate-specific lectin which recognises activated homologous opsonins

Summary Haemocytes of the gastropod mollusc, Helix pomatia, possess on their surface a membrane-integrated GalNac-specific lectin which binds to and stimulates phagocytosis of GalNac-bearing target cells (human A erythrocytes) only in the presence of extracellular calcium ions. Target cells without GalNac moieties on their surface (human B and bovine erythrocytes) are not recognised. Helix haemocytes also possess a Ca²⁺-independent mannose-6-phosphate-specific lectin on their surface which, in the absence of extracellular calcium ions, enables recognition and phagocytosis of A rbc opsonised with agglutinins isolated from either the snail's albumin gland or serum. These opsonins, however, bind to host haemocytes only after binding to GalNac moieties on the surface of test particles. Our results indicate that such a ligand-specific opsonin/target cell interaction apparently induces a conformational change in the opsonin, resulting in exposure of mannose 6-phosphate moieties that are recognised by the Ca²⁺-independent lectin on the surface of the haemocytes.Abbreviations BSA bovine serum albumin - bv bovine - DAB 3-3-diamino-benzidine tetrahydrochloride - ELISA enzyme-linked immunosorbent assay - GalNac N-Acetyl-D-galactosamine - G6-P glucose 6-phosphate - HE haemocyte extract - HPA Helix pomatia albumin gland agglutinin - HPA PO peroxidase-labelled HPA - M6-P mannose 6-phosphate - ML monolayer - MLS monolayer supernatant - OPD orthophenylene diamine - PBS phosphate buffered saline - PMSF phenylmethylsulphonyl fluoride - PO peroxidase - rbc red blood cells - RT room temperature - SA Helix pomatia serum agglutinin - TBS Tris buffered saline     

Glial patterning during postembryonic development of central neuropiles in the brain of the honeybee

 Glial cells are involved in several functions during the development of the nervous system. To understand potential glial contributions to neuropile formation, we examined the cellular pattern of glia during the development of the mushroom body, antennal lobe and central complex in the brain of the honeybee. Using an antibody against the glial-specific repo-protein of Drosophila, the location of the glial somata was detected in the larval and pupal brain of the bee. In the early larva, a continuous layer of glial cell bodies defines the boundaries of all growing neuropiles. Initially, the neuropiles develop in the absence of any intrinsic glial somata. In a secondary process, glial cells migrate into defined locations in the neuropiles. The corresponding increase in the number of neuropile-associated glial cells is most likely due to massive immigrations of glial cells from the cell body rind using neuronal fibres as guidance cues. The combined data from the three brain regions suggest that glial cells can prepattern the neuropilar boundaries. Received: 3 November 1996 / Accepted: 7 February 1997