tracts and synaptic centres resulting from metamorphic re-differentiation of larval neurons is phagocytosed by neuropil-associated glial cells. Phagocytic glial ...
Dev Genes Evol (1996) 206:277–280
© Springer-Verlag 1996
S H O R T C O M M U N I C AT I O N
&roles:Rafael Cantera · Gerhard M. Technau
Glial cells phagocytose neuronal debris during the metamorphosis of the central nervous system in Drosophila melanogaster
&misc:Received: 23 January 1996 / Accepted in revised form: 21 May 1996
&p.1:Abstract Using electron microscopy we demonstrate that degenerating neurons and cellular debris resulting from neuronal reorganization are phagocytosed by glial cells in the brain and nerve cord of the fruitfly Drosophila melanogaster during the first few hours following pupariation. At this stage several classes of glial cells appear to be engaged in intense phagocytosis. In the cell body rind, neuronal cell bodies are engulfed and phagocytosed by the same glial cells that enwrap healthy neurons in this region. In the neuropil, cellular debris in tracts and synaptic centres resulting from metamorphic re-differentiation of larval neurons is phagocytosed by neuropil-associated glial cells. Phagocytic glial cells are hypertrophied, produce large amounts of lysosome-like bodies and contain a large number of mitochondria, condensed chromatin bodies, membranes and other remains from neuronal degeneration in phagosomes. &kwd:Key words Nervous system development · Metamorphosis · Phagocytosis · Glial cells · Drosophila melanogaster&bdy:
Introduction During metamorphosis of the central nervous system (CNS) in the fruitfly Drosophila melanogaster, many larval neurons are integrated into adult networks through a process that requires a substantial degeneration of larval arborizations prior to the differentiation of adult branches (reviewed by Truman 1990; Truman et al. 1992). In the corpora pedunculata for example, one of the most important brain centres, the differentiation of Edited by J. Campus-Ortega R. Cantera (✉) Department of Zoology, Stockholm University, S-106 91 Stockholm, Sweden G.M. Technau Institut für Genetik-Zellbiologie, Saarstrasse 21, D-55122 Mainz, Germany&/fn-block:
adult arborizations is preceded by a massive degeneration of larval axons and dendrites occurring during the hours following pupariation (Technau and Heisenberg 1982). Another major mechanism contributing to the development of the CNS is the elimination of certain neurons by programmed cell death. In the fruitfly, central neurons are eliminated by programmed cell death both during the embryonic (Abrams et al. 1993; White et al. 1994; Sonnenfeld and Jacobs 1995a; White and Steller 1995; Zhou et al. 1995) and postembryonic (Fischbach and Technau 1984; Kimura and Truman 1990; Truman 1990) phases of development. Metamorphosis thus involves at least two types of neuronal degeneration in the CNS of the fly: entire cells are eliminated by programmed cell death and parts of cells are eliminated during postembryonic re-differentiation. As for other major morphogenetic processes, cell death and cell differentiation are supposed to require several interactions between particular types of cells. An interesting aspect in the study of these cell interactions is phagocytosis, the mechanism by which the cellular debris resulting from programmed cell death and other degenerative processes is cleared out from the tissue. In the embryonic Drosophila CNS, apoptotic cells are phagocytosed by glial cells and haemocytes (Zhou et al. 1995; Sonnenfeld and Jacobs 1995b). The determination of which cells phagocytose neuronal debris in the metamorphosing CNS was the goal of the present investigation.
Materials and methods Wild-type fruit flies (Berlin and Canton S strains), Drosophila melanogaster, reared on standard conditions were used. Animals staged at pupariation (“white pupa” stage, 0 h after puparium formation – APF) and kept at 25° C. At the desired stage the CNS (brain and nerve cord) was dissected in saline and fixed immediately in a mixture containing 4% paraformaldehyde, 2.5% glutaraldehyde and 2.5% tannic acid in 0.1 M phosphate buffer, pH 7.2. After several rinsings in phosphate buffer the tissue was postfixed in 1% osmium tetroxide for 1 h, rinsed again and dehydrated in ethanol for embedding in Agar resin (Agar Scientific Ltd.). Semithin sections (2 µm in thickness) were stained in boracic toluidine
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Fig. 1A–E Phagocytosis of neuronal debris in the nerve cord of Drosophila during the first hours after pupariation. A Transverse section across abdominal neuromere 4 (2-µm section stained with toluidine blue) showing an overview of the distribution of phagocytic sites in the nerve cord detailed in the following electron micrographs. The arrows point at sites of phagocytosis where degenerating neuronal cell bodies are engulfed by different types of glial cells: 1 Subperineurial glia, 2 cell body glia, 3 interface glia The arrowheads point at sites of phagocytic activity in dark patches of cytoplasm from enlarged glial extensions penetrating the neuropil (the ultrastructural details of one of these sites are shown in Fig. 1E). Notice that the nerve cord is surrounded by a complete basal lamina (BL) without attached haemocytes at this stage (Scale bar 20 µm). B Beneath the basal lamina (BL) and perineurium (Pe), a
subperineurial glia engulfs a large phagosome containing a degraded neuronal cell body (DN). Note that the same glial cell covers the cell bodies of normal looking neurons (arrows). C A cell body glia surrounding several neuronal cell bodies (arrows) encloses a large phagosome containing a degraded cell body (DN). D An interface glial cell (Nu nucleus) loaded with lysosome-like organelles, lamellate bodies (LB) and phagosomes fully encloses a fascicle of healthy axons (Ax) in a ventral longitudinal connective. Two degenerating axons engulfed in phagosomes are marked by arrows. E Deep in the neuropil (a site corresponding with those marked by arrowheads in 1A) an enlarged extension from a phagocytic glial cell contains lamellate bodies (LB) and phagosomes (asterisk), some of which contain axons (Ax). Magnification: B ×6600, C ×6300, D ×13800, E ×8800&ig.c:/f
blue and mounted in Permount as a reference for the ultrastructural study and to ascertain if haemocytes were associated with the CNS at this stage. Complete series of serial histological sections were prepared from five pupal CNS. Ultra-thin sections of selected areas were mounted on copper grids, counterstained for 50 min in 3% uranyl acetate and viewed in a JEOL 100CX electron microscope.
Results and Discussion With the aid of electron microscopy we demonstrate here that cellular debris resulting from degenerative processes occurring during the metamorphosis of the fly central
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nervous system (CNS) are phagocytosed by glial cells. Phagocytosis was detected throughout the CNS during the first hours following pupariation. Figure 1A shows an overview of the histological appearance of the nerve cord at this stage. At this low magnification, sites of phagocytosis by different classes of glial cells (we follow the classification of glial cells proposed by Ito et al., 1995) are indicated and the ultrastructural details are shown in the following figures. All glial cells beneath the perineurium appear to be engaged in intense phagocytosis with the exception of the midline glial cells. Phagocytic glial cells are hypertrophied, produce large amounts of lysosome-like bodies and contain many mitochondria, condensed chromatin bodies, lamellate bodies and other debris most likely resulting from neuronal degeneration in phagosomes (Fig. 1 and 2). In the cell body rind, whole cell bodies from neurons in different stages of degeneration were found to be completely engulfed by the same glial cells that also form extensive cytoplasmic processes enveloping healthy-looking neurons. Cell bodies located in the periphery are thus engulfed by subperineurial glia (Fig. 1B), and those located more deeply are engulfed by cell body glia (Fig. 1C). Cell bodies degenerating in the vicinity of the neuropil can also be phagocytosed by glial cells at the cortex/neuropil interface (interface glia). The examples in Fig. 1B and C also show that the same glial cells extending the typical electron-dense cytoplasmic prolongations between neuronal cell bodies also contain phagosomes with organelles, chromatin and other cellular debris. Neuropil-associated glia send processes into the neuropil, where we found them to phagocytose neuronal debris in tracts and synaptic centres of the nerve cord and brain. In the nerve cord, the neuropil is loaded with phagocytic processes from the interface glial cells (seen as dark patches in the histological overview shown in Fig. 1A). Figure 1D shows an example of a phagocytic interface glia surrounding longitudinal axons in the ventral connective. Prolongations from this type of glial cell enter deeply into the neuropil where they form enlarged areas of phagocytic activity. In these glial prolongations we found phagosomes, some containing degenerating axons, lysosome-like bodies, and lamellate bodies (Fig. 1E). Figure 2 shows examples of other neuropil-associated glial cells engaged in phagocytosis around and within the peduncles of the corpora pedunculata in the brain. It is important to remember that the present study concerns only a relatively short period in metamorphosis, since the tissue examined under the electron microscope was prepared exclusively from animals a few hours after pupariation. During embryonic development, the apoptotic corpses of midline glial cells have recently been reported to be extruded from the nerve cord and thereafter phagocytosed by haemocytes (Zhou et al. 1995). According to the detailed observations on programmed cell death during Drosophila embryogenesis provided by Abrams et al. (1993), phagocytosis of CNS cells by a haemocytes is probably restricted to early embryonic stages, or perhaps even particular cell progenies.
Fig. 2A–D Electron microscopic images of phagocytosis by glial cells in the brain at 7 h after pupanium formation (APF). A Overview of pupal brain neuropil with a neuropil glial cell (Gl) at the rim of the mushroom body peduncle (Pe, seen in cross section) in the anterior protocerebrum. Magnification ×2000. B At higher magnification (×12500) a phagosome (Ph) and a lysosome-like (Ly) organelle containing a membranous structure are clearly seen in this cell. C A neuropil glial cell (Gl) associated with the calyx (Ca) of the mushroom body is loaded with lysosome-like organelles and phagosomes (some are highlighted by arrows) containing membranes, microtubules and other cell debris at different stages of degradation. Magnification ×12500
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According to these authors, phagocytic haemocytes engulf dying cells in the early embryonic nerve cord, but ...“do not invade the tightly packed cell body layer”... from stage 16. More recently, Sonnenfeld and Jacobs (1995b) confirmed that haemocytes phagocytosed apoptotic neurons immediately outside the embryonic nerve cord and, in addition, presented data showing that phagocytosis was initiated inside the nerve cord by glial cells, which thereafter would release this material outside the nerve cord for further phagocytosis by haemocytes. In this study, we paid special attention to the possibility that haemocyte phagocytosis will also occur during early metamorphosis. However, after careful examination of serial sections we found that haemocytes associated with the surface of the early pupal CNS are very rare and we did not find haemocytes of any type inside the perineurial sheath of the CNS. Thus, the results reported here indicate that phagocytosis of neuronal material in the brain and nerve cord during early metamorphosis is performed exclusively by glial cells. &p.2:Acknowledgements We would like to express our gratitude to Prof. B. Afzelius (Stockholm) for his support and stimulating discussions during the preparation of this study and Drs. I. Mayer and D. Hultmark for comments on the manuscript. Part of the work was supported by a grant from the Deutsche Forschungsgemeinschaft to G.M.T.
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