development and function of embryonic central nervous system glial cells indrosophila

DEVELOPMENTAL GENETICS 18:40-49 (1996)

REVIEW ARTICLE

Development and Function of Embryonic Central Nervous System Glial Cells in Drosophila CHRISTIAN KLAMBT, THOMAS HUMMEL, THOMAS MENNE, EVELIN SADLOWSKI, HENRIKE SCHOLZ, AND ANGELIKA STOLLEWERK Znstitut fur Entwicklungsbiologie, Universitat zu K d n , Koln, Germany

ABSTRACT Each abdominal neuromere of a Drosophila embryo contains about 60 glial cells [Klambt C, Goodman CS (1991): Glia 4:205-213; Ito ef a/. (1995): Roux’s Arch Dev Biol, 204:284- 3071. Among these, the midline and longitudinal glia are described to some detail. The midline glia are located dorsally in the nerve cord ensheathing the two segmental cornmissures. They are required for the proper establishment of commissures. The longitudinal glia, the A and B glia, and the segment boundary cells (SBC) are covering the longitudinal connectives. The longitudinal glia prefigure longitudinal axon paths and appear capable of regulating the expression of neuronal antigens. In the following we summarize the knowledge on the function of these glial cells.

Key words: Drosophila, g lia, neu ron-g lia interaction

o 1996 WiIey-Liss, Inc.

INTRODUCTION Any complex nervous system of vertebrate or inver-

tebrate origin mainly consists of two different cell types: neurons, which send out axonal processes to form the intricate neuronal lattice, and glial cells, which are found intermingled between. Initially glial cells were considered as “Kitt” or glue material by Vir- chow in the middle of the 19th century. However, since then an increasing number of different functions have been attributed to them, such as electrical insulation and ionic homeostasis, nutrition, and control of neuronal cell proliferation and survival (which can be a reciprocal interaction) [Barres, 1991; Masu et al., 1993; Ebens et al., 1993; Buchanan and Benzer, 1993; Rey- nold and Woolf, 1993; Xiong and Montell, 1995; Auld et al., 19951. An additional role of glial cells is seen during growth cone guidance, which in part depends on direct cell-cell contact. Extensive studies have shown the role of glia and other nonneuronal cells during neu-

ronal development, both as a permissive, and some- times as an active substrate for migrating neurons and extending growth cones [Singer et al., 1979; Rakic, 1971, 1972; Silver et al., 1982; Jacobs and Goodman, 1989a,b; Fishell and Hatten, 1991; Garriga et al., 1993; Gorczyca et al., 19941. However, despite the wealth of well-described developmental phenomena, little is known about the mechanisms and molecules underlying glial differentiation or glial-neuronal interactions.

Using conventional histological methods, a variety of glial cells have been described in insects [Wiggles- worth, 1959; Carlson and Saint Marie, 1990; Hoyle, 1986: Cantera, 19931. An even higher, quite remark- able degree of glial diversity has been revealed in Dro- sophila with the help of the enhancer trap methodology [Klambt and Goodman, 1991; Nelson and Laughon, 1993; Ito et al., 19951. The introduction of this lineage marker technique in Drosophila [O’Kane and Gehring, 1987; Bier et al., 1989; Bellen et al., 19891 has lead to the isolation of a number of fly strains which express the lacZ reporter gene specifically in a limited number of glial cells. Most importantly, these new lineage markers can serve as tools to selectively study individual glial cells not only in wild-type, but also in different mutant backgrounds [Klambt et ad., 1991; Giangrande et al., 1993; Giangrande, 1994, 1995; Winberg et al., 1992; Jacobs, 1993; Choi and Benzer, 1994; Klaes et al., 1994; Sonnenfeld and Jacobs, 1994; Xiong and Montell, 19951.

Work from a number of labs has demonstrated that central nervous system (CNS) glial cells of Drosophila can arise from different origins: the perineurial sheath cell layer (which generates the extracellular neural

Received for publication May 5, 1995; accepted June 15, 1995.

Address reprint requests to Christian Klambt, Institut fur Entwick- lungsbiologie, Universitat zu Kiiln, D-50923 Koln, Germany.

0 1996 WILEY-LISS, INC.

EMBRYONIC CNS GLIAL CELLS IN DROSOPHILA 41

lamella ensheathing the entire CNS) is of mesodermal origin [Edwards et al., 19931, the midline glial cells develop from a special set of mesectodermal progenitors [Klambt et al., 1991; Bossing and Technau, 19941, the longitudinal glial cells stem from a lateral glioblast [Jacobs et al., 19891, and some glia such as the A and B glia are progeny of neuroblasts which generate mixed lineages [Udolph et al., 19931.

Regardless from their origin, a common feature of glia is their early appearance in the nervous system, often prefiguring axonal pathways. In recent years, studies have begun to focus on the early events during the pioneering of the first axon tracts, and in particular on the substrates along which these initial pathways form. An excellent demonstration of the function of glial cells during growth cone guidance was obtained in the grasshopper nervous system. Growth cones which normally leave the CNS to pioneer the intersegmental nerve root make a characteristic pathway choice at a specific glial cell, the segment boundary cell (SBC). After ablation of this cell the growth cones fail to exert the correct pathway choice, showing that the SB cell provides important directional guidance cues [Bastiani and Goodman, 19861.

In the developing CNS of Drosophila, glial cells have been implicated in the formation of a number of axon pathways based on electron microscopic studies [Jacobs and Goodman, 1989a,bl and more recently, mutational analysis has provided further evidence for this function [Klambt et al., 1991; Seeger et al., 1993; Gorczyca et al., 1994; Klaes et al., 19941. In the following paper we want to review two aspects of glial cell research. First we summarize glial cell function in the CNS, focusing on midline and longitudinal glial cells, and second we summarize the molecular mechanisms underlying glial cell differentiation. To what extent do glial substrates determine axonal pattern formation in the developing CNS? And how do neuronal growth cones interact with a glial prepattern in deciding which way to grow?

MIDLINE GLIA Development of the Midline

The midline of the insect nervous system comprises a small group of specialized morphologically distinct cells also called mesectodermal cells [Hatschek, 1877; Escherich, 1902; Poulson, 19501. Similar to their vertebrate counterparts, the floor plate cells, the midline cells of the Drosophila CNS are among the first CNS cells to be specified, as revealed by the specific expression of the gene single minded [Crews et al., 1988; Thomas et al., 1988; Schoenwolf and Smith, 19901. Again similar to the development of the vertebrate floor plate, which requires an inductive signal originating from the notochord, mesectodermal specification in Drosophila requires inductive signaling from the mesoderm [Leptin and Roth, 19941. Interestingly, during later development the midline exerts inductive

influences on neighboring ectodermal cells [Kim and Crews, 19931.

As the germ band elongates during gastrulation, all midline progenitor cells delaminate into the same layer as the neuroblasts. Using enhancer trap marker lines and DiI labeling techniques seven to eight midline progenitor cells have been found per segment, which divide one or two times to yield about 26 midline cells per abdominal neuromere [Klambt et al., 1991; Bossing and Technau, 19941. The anteriormost three midline progenitors cells give rise to three pairs of midline glial cells (MGA, MGM, and MGP) [Jacobs and Goodman, 1989; Klambt et al., 19911. This is in stark contrast to the grasshopper CNS, where midline glial cells are generated by the median neuroblast [Condron and Zinn, 19941. The production of different cell types by this stem cell is controlled by the activity of engrailed and PK-A [Condron et al., 1994: Condron and Zinn, 19951. In Drosophila, the remaining midline progenitor cells generate a set of midline neurons (two MP1 neurons, six ventral unpaired median [VUMI neurons, two dorsal unpaired median [DUM] neurons) as well as the median neuroblast [Klambt et al., 1991; Bossing and Technau, 19941.

Development of Midline Glial Cells Early specification of midline glial cells can be fol-

lowed by the expression of P-galactosidase in a number of enhancer trap lines during germband extension. At this developmental stage the midline glia are elon- gated cells with their basal side anchored in the ventral epidermis, and the nucleus located dorsal close to the apical membrane. In a 12-hour-old Drosophila embryo, the six midline glial cells have lost their connections to the epidermis and lie in front of, between, and just behind the two axon commissures, respectively [Jacobs and Goodman, 1989a; Klambt et al., 19911. In 16 to 17-hour-old embryos only two to four midline glial cells enwrapping the commissural axons are found per neuromere [Bossing and Technau, 1994; Fig. 11. This difference in the number of midline glial cells is likely to be due to programmed cell death, as the number of midline glial cells is increased in mutations which block apoptosis [Sonnenfeld and Jacobs, 19951. The exact mechanisms regulating midline glia cell number are not understood to date.

At the end of embryonic development the midline glia further subdivide both anterior and posterior commissures into three major compartments which are maintained throughout larval stages. During larval life, the midline glia undergo endomitotic DNA replication [Prokop and Technau, 19941 and their nuclei grow considerably in size [Stollewerk et al., 19951. Dur- ing metamorphosis, the giant glial cell nuclei disappear and tight clusters of much smaller nuclei can be detected instead, suggesting cell fragmentation. The resulting cell fragments are predicted to be nonviable and should be cleared from the tissue within a short

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time. In agreement with this, no midline glial cells can be detected in older pupae [Stollewerk et al., 19951. In this respect it is interesting to note that during vertebrate embryogenesis glial cells are formed in the floor plate, but seem to disappear from the midline during the first postnatal week [McKanna, 19931.

Function of Midline Glial Cells First thoughts on the function of the CNS midline

cells in insects date back more than 100 years, when the midline cells were first recognized as a discrete set of cells within the CNS (then called Mittelstrang- zellen) and were believed to be important for the formation of commissural connections within a neuromere [Hatschek, 1877; Escherich, 19021. More recent work has brought alive this old hypothesis and allowed the assignment of unique functions to individual midline cells during the establishment of the axonal scaffold. In Drosophila the formation of the two segmental axon commissures takes place during embryonic stage 12 and occurs in a series of discrete steps [Klambt et al., 19911. The first CNS growth cones are detected at about 8.5 hours of development and show directed growth straight toward a specific VUM neuron at the midline. At this developmental time point midline glial cells are found one cell diameter anterior to the place where commissural axons cross the midline and it thus appears unlikely that they play a role in the initial development of the posterior commissure. Once the posterior commissure is formed, the midline glia start to migrate posteriorly and come into a position where they might serve as attractive cues for later commissural growth cones establishing the anterior commissure [Klambt et al., 19911. In this respect it is interesting to note that the gene commissureless (comm), which leads to a loss of commissures when removed, is expressed in the midline glia [Seeger et al., 1993; Tear et al., in preparation, C.S. Goodman, personal communication]. comm encodes a small, unusual transmembrane protein, but may not be an attractive guidance molecule by itself, since, although some protein may be secreted most of it appears sequestered in the Golgi [Tear et al., in preparation, C.S. Goodman, personal communication]. However, a Drosophila homolog of the unc6lnetrin family of genes, which orient the path of commissural growth cones in Caenorhabditis elegans and vertebrates [Hamelin et al., 1993; Serafini et al., 1994; Kennedy et al., 19941, has been recently cloned and is also expressed by midline glia [Mitchell et al., 19941.

Once the two segmental commissures have been established, two of the four midline glial cells start to crawl along the VUM growth cones in-between the anterior and posterior commissures, pushing them into their mature ladder-like appearance [Klambt et al., 1991; Figs. 2A, 3; see also Choi and Benzer, 1994 who describe migration of glial cells along photoreceptor axons]. Thus, the development of the axonal pattern in

the CNS requires a series of sequential neuron-glia interactions which appear necessary for the formation and the later separation of commissures. This later glial function is also evident from the analysis of a growing number of mutant phenotypes [Klambt et al., 1991; Raz and Shilo, 1992; Rutledge et al., 1992; Klambt, 1993; Kolodkin et al., 1994; Anderson et al., 1995; Hummel et al., in preparation].

A class of mutants [sim, sichel (sic), spit2 (spi), Star (S), rhomboid {rho), and pointed (pnt)l, classified as the spitz group based on cuticle defects a t the ventral midline, also affects the development of the CNS midline [Mayer and Nusslein-Volhard, 1988; Klambt et al., 1991; Klambt, 1993; Sonnenfeld and Jacobs, 19941. Furthermore, mutations in spi, S, rho, and pnt lead to a similar embryonic CNS phenotype consisting of fused commissures. The initial development of the axon commissures appears normal; however, the mutant phenotypes appear at, the stage when midline glia should normally migrate in-between the two commissures, resulting in their separation. Instead of separating into the distinct anterior and posterior cornmissures, these axon tracts stay close together in the mutants. The midline glial cells initially develop normally but then fail to migrate and finally die (Fig. 2B).

Molecular Specification of Midline Glial Cells Early midline specification can be monitored by fol-

lowing the expression of single minded (sim). sim encodes a basic helix-loop-helix (bHLH) type transcription factor and regulates the expression of most genes that are subsequently activated in the midline. Loss of function alleles of the sim gene lead to a selective death of all midline cells after gastrulation [Klambt et al., 1991; Nambu et al., 1990, 19911. Expression of sim is controlled in part by inductive influences originating from the mesoderm, which might be mediated by Notch and Delta [Menne and Klambt, 1994; Martin-Bermudo et al., 1995; see Hassan and Vaessin, this issue for de- scription of neurogenic genes]. Notch-mediated signaling appears not only to be required for the specification of the mesectodermal anlage, but also for the determination of midline glia cell fate within the mesectoderm, since removal of zygotic Notch function leads to a com- plete loss of midline glial cells [Menne and Klambt, 19941.

sim might have a specific function during midline glia development. Although szm is initially expressed in all midline progenitors, it becomes highly restricted to the midline glial cells [Crews et al., 19881. We have tested whether the accumulation of high levels of sim protein determines midline glia development by over- expression of s i n in the midline. However, the expression of high levels of sim in all midline cells does not appear to alter cell fate decisions at the midline [Menne and Klambt, in preparation].

Clues to the molecular mechanisms which govern midline glial development stem from mutations which

44 KLAMBT ET AL. affect midline glial development more specifically. Analysis of a number of mutants revealed that the epidermal growth factor (EGF)-receptor mediated ras signaling cascade is crucial for proper midline glia development. In the embryonic nervous system, the EGF-receptor tyrosine kinase (DER) is expressed specifically in the midline glial cells [Zak et al., 19901. Removal of DER function in the midline glia using a temperature-sensitive DER allele demonstrated the absolute requirement of EGF-receptor-mediated signaling for midline glial development [Raz and Shilo, 19921. Not surprisingly, mutations in the gene spi, which encodes a Drosophila homolog of transforming growth factor alpha (TGF-a), a ligand of the EGF-re- eeptor, result in the same midline glia phenotype [Klambt et al., 1991; Rutledge et al., 19921. Similarly, mutations in the genes S and rho, which both encode transmembrane proteins implicated in DER signaling, lead to a disruption of midline glia function [Klambt et al., 1991; Bier et al., 1990; Sturtevant et al., 1993; Kolodkin et al., 19941.

The signal transduction cascade initiated by binding of a ligand to a receptor tyrosine kinase (RTK) has been conserved throughout evolution [Dickson and Hafen, 19941. A common biochemical pathway couples RTK phosphorylation via ras to a cascade of serinekhreo- nine kinases (raL MEK, MAPK), which results in the phosphorylation of specific transcription factors and thus mediates the signal transfer from the cell membrane to the nucleus. The general conservation of this signaling pathway and the fact that in addition to DER, a nuclear target of RTK signaling encoded by pntP2 (see below), is expressed in the midline glia, makes it likely that RTK signaling will direct midline glia differentiation. Indeed, all members of this signaling cascade tested to date are expressed in the midline glia (Fig. 3). Furthermore, mutations in drk, which encodes an SH3-SH2-SH3 adapter protein [see Dickson and Hafen, 1994 for review], also result in a fused commissure phenotype indicative for midline glia defects (H.S., unpublished).

The RTK-mediated signaling cascade finally directs the execution of a specific developmental program by the activation of specific transcription factors. One such example is seen in the gene pnt, which encodes two ETS domain transcription factors, P1 and P2. In the embryonic CNS, pnt is expressed specifically in glial cells and mutations in pnt lead to a similar fused commissure phenotype as mutations in the EGF-receptor [Klambt, 19931. In addition, pnt is required during photoreceptor cell development in the eye imaginal disc, where it acts downstream of the sevenless RTK signaling cascade [Brunner et al., 1994; O’Neill et al., 19941. The pntP2 protein is expressed in the midline glial cells and contains a second protein domain conserved during evolution, called the pointed domain. This region harbors a MAP-kinase phosphorylation site which can be phosphorylated by MAP-kinase in

uitro and is required for normal pnt function in viuo [Brunner et al., 1994; O’Neill et al., 19941. Thus, pnt appears to act a t the end of the RTK signaling cascade. Interestingly, the midline glia phenotype associated with pnt is slightly different to that seen in spi or DER mutant embryos. In pnt embryos, midline glial cells initially differentiate and even appear to migrate. But instead of following growth cones of the VUM neurons, the midline glia migrate along commissural axon tracts toward the longitudinal connectives. The differ- ences in glial phenotypes suggest that there are branch points in the DER signaling cascade which allow the control of multiple target proteins involved in glial development.

Other transcription factors, like the POU-domain transcription factor encoded by the gene drifter [Ander- son et al., 19951 and the Zn-finger type transcription factor encoded by tramtrack [S. Harrison, personal communicationl, are expressed in the midline glia but their connection to DER signaling still needs to be determined. These transcription factors are likely to control the expression of a number of different genes like argos and slit. argos is expressed specifically in four midline glial cells and encodes a secreted protein containing a single EGF-like domain. During later development, argos is expressed in the eye imaginal disc, where it acts in a non cell autonomous manner as a negative regulator of cell fate decisions. However, although amorphic argos alleles are associated with embryonic lethality, no phenotype can be seen in the midline glia [Freeman et al., 19921. This might indicate that argos function is not required for midline glial development or that additional gene functions can in part compensate for the effects caused by the loss of argos. The latter possibility appears true, since argos is flanked by at least two closely interacting genes [Wem- mer and Klambt, 19951. slit expression is found initially in all midline cells but becomes restricted to midline glia during later development. Loss of slit, which encodes a protein with multiple leucine-rich repeat (LRR) and EGF-like domains, leads to degeneration of all midline cells. slit protein is secreted and transferred

Fig. 2. Function of CNS glial cells. A,B: Frontal views of dissected stage 16 embryos (D, stage 14) carrying the AA142 enhancer trap insertion which directs P-galactosidase expression in the midline glial cells (blue). The axonal scaffold is visualized by MAb BP102 and sub- sequent HRP immunohistochemistry (brown). A In a wild-type embryo, anterior and posterior commissures are separated by midline glial cells. The midline glia can be seen in contact with VUM axons (arrowheads). B: In a mutant spi embryo, the midline glia fail to migrate in-between anterior and posterior commissures which conse- quently appear fused. In some segmenk motoneurons flanking the midline appear trapped between the commissures (arrow). C: Sche- matic drawing showing the cell transplantation experiment used to demonstrate inductive capabilities of glial-like cells. D: A pntPl expressing cell clone (brown) is inducing 22C10 expression in the mesoderm,

c HRP labelled, pntPl expressing donor cell

/-

unlabelled wild-type host

Fig. 2 A-D.

46 KLAMBT ET AL.

spitz (T P or) from the midline glia to most of the CNS axons [Roth- berg et al., 1988, 19901.

Interestingly, the very same signal transduction cascade which appears to control glia development in the embryonic midline controls neuronal cell fate decisions in the developing compound eye. But where does spec- ificity come about? Why does a recipient cell develop as a glial cell instead of as a neuronal cell in response to the activation of the same transcription factor by a common signal transduction cascade? The answers to these questions will require characterization of further genes involved in midline glial development. The fused commissure phenotype displayed by embryos lacking functional midline glial cells should facilitate the iden- tification of such genes in mutant screens [Seeger et al., 1993; Hummel et al., unpublished].

w

midine glia

Development of Longitudinal Glial Cells Longitudinal glial cells were first described in the

grasshopper embryo [Bastiani and Goodman, 19861. Observations on their migration and division patterns suggested that this type of glia might arise from a single transient, lateral glioblast per hemineuromere. In Drosophila, the existence of a lateral glioblast was first revealed by analyzing the expression pattern of the gene fushi taruzu (f tz) [Doe et al., 19881. Using f tz promoter-lac2 fusion constructs as lineage tracers, six longitudinal glial cells were counted as progeny of the lateral glioblast [Jacobs et al., 1989; Fig. 1Dl. Interest- ingly, lac2 reporter constructs utilizing the DNA-binding sites for the homeobox proteins f tz or engrailed (en), also direct lac2 expression in embryonic glia [Vincent et al., 1990; Nelson and Laughon, 19931, which however is not dependent on either f tz or en. Nevertheless, mutational analysis has shown that the homeo domain binding DNA core sequence 5’AATTA3’ is required for this glial-specific expression, suggesting that other homeobox proteins are likely to be responsible for the generation of this expression pattern [Vincent et al., 19901. A candidate gene encoding such a protein is the recently isolated glia-specific gene rep0 [Xiong et al., 1994; Campbell et al., 1994; Halter et al., 19951.

In contrast to neuroblasts which typically divide asymmetrically into a smaller ganglion mother cell and a large neuroblast, the lateral glioblast divides symmetrically to produce two glial cells which leave the neuroblast layer and migrate medially [Jacobs et al., 1989; Halter et al., 19951. Once these two longitudinal glial cells have reached the aCC neuron they divide once more in a symmetric fashion. As the longitudinal connectives form during stages 13-15, a t least one further division occurs. The final number of longitudinal glial cells is eight. The exact lineage relationship of the embryonic longitudinal glial cells 7 and 8 is unknown to date. During larval life these glial cells undergo endomitotic DNA replication cycles [Prokop and Technau, 19941.

midline neuron Fig. 3. DER signaling during midline glia development. Schematic

sagittal drawing of the events which control differentiation of midline glial cells. Genes expressed in the midline glia are indicated. The Drosophilu homolog of the EGF-receptor is expressed in the midline glial cells. Binding of a ligand leads to dimerization and autophos- phorylation. Binding of the drk SH3-SH2-SH3 adapter protein leads to the activation of ras, which in turn is able to activate ruf, also known as MAP-kinase kinase kinase (MAPKKK). Through another kinase, the activity of the MAP-kinase, which is encoded by the gene rolled frZ), is controlled, which in turn is able to directly phosphory- late transcription factors such as pointedP2. The activity of pnt controls the glial migration along cell processes of the VUM neurons.

Longitudinal Glial Cell Function The longitudinal glial cells are present early during

development and prefigure the longitudinal axonal connectives. Extensive electron microscopic studies have first suggested that the longitudinal glia are indeed used as a substrate during the extension of the first longitudinal axons [Jacobs and Goodman, 1989bl. Further evidence that the glia scaffold prefigures longitudinal axon tracts stems from genetic analyses [Ja- cobs, 1993; Klaes et al., 19941. In this respect it is interesting to note that expression of a neuronal protein labeled by the monoclonal antibody (MAb) 22C10 on specific neurons depends on functional glia. Further- more, following cell transplantation, CNS glial cells are capable of inducing expression of the MAb 22C10 antigen in neighboring cells [Klaes et al., 1994; Fig. 2C, D, 41. Thus, neuron-glia interaction might in part directly control axonal pathfinding by regulating the set of membrane proteins expressed by a given neuron. The dependence of Mab 22C10 antigen expression on glia now allows for the first time to genetically screen for genes required for neuron-glia communication (see Fig. 4).

Additional functions of CNS glial cells are deduced from studying the rep0 mutant phenotype. rep0 is expressed exclusively in glial cells and embryos lacking rep0 function display fasciculation defects as well as


Fig. 4. Neuron-glia interaction. Schematic frontal view of the neuron-glia interaction controlling neuronal antigen expression. Differ- entiation of glia cells (ovals) is in large part controlled by pnt. Through an unknown signaling mechanism, pnt controls neuron-glia cross talk, which leads to the expression of the MAb 22C10 antigen (black dots) on,neighboring neurons. The nature of the MAb 22ClO antigen is presently unknown. To unravel the genes which are involved in this interaction we are undertaking a large-scale mutant screen for mutations which effect expression of the MAb 22C10 antigen on these neurons.

they fail to undergo proper condensation of the ventral nerve cord [Campbell et al., 1994; Halter et al., 19951. A similar failure in nerve cord condensation is also ob- served in mutant prospero embryos, where also defective glial cells are belived to be responsible for this phenotype [Doe et al., 19911. A further, yet untested role of embryonic glial cells might be to provide neurotrophic factors for surrounding neurons. This is suggested by the phenotype associated with hypornorphic rep0 alleles, which display an age-dependent neuronal degeneration [Xiong and Montell, 19951. Similarly, phenotypic analysis of mutations in the gene drop- dead suggests that normal glia might prevent brain degeneration, perhaps by providing the appropriate neurotrophic factors [Buchanan and Benzer, 19931. Finally, glial cells ensheath the CNS and provide a blood-brain barrier to ensure that neurons are bathed in the appropriate ionic environment [Auld et al., 19951.

Molecular Specification of Longitudinal Glial Cells

Genes which affect the specification of lateral glioblast have not yet been described, prospero encodes a large nuclear protein with a distantly related homeobox and is expressed in the glioblast and its progeny [Doe et al., 19911. Embryos lacking prospero func-

tion still contain glial cells which, however, do not differentiate properly [Jacobs, 19931. The severe axonal outgrowth defects seen in mutant prospero embryos cannot be solely attributed to glia malfunction, since the gene is also expressed in neuronal cells. Other transcription factors known to be expressed in the longitudinal glial cells are rep0 and pnt . Lack of r e p does not lead to any profound axonal pattern defect but loss of pnt results in glial differentiation defects as well as in axonal pattern defects [Klambt, 1993; Klaes et al., 1994; Xiong et al., 1994; Campbell et al., 1994; Halter et al., 19951. In the embryonic CNS, both genes are expressed exclusively in glial cells. The longitudinal glial cells express rep0 and the pntPl form, in contrast to the midline glia, which express the pntP2 form but not repo.pntP1 and pntP2 share the DNA-binding domain, but pntPl lacks a MAP-kinase phosphorylation con- sensus sequence. Unlike the pntP2 form, which requires phosphorylation to become an active transcription factor, pntP1 acts as a strong transcription activator without any additional activation [Klaes et al., 1994; O’Neill et al., 19941. When expressed ectopi- cally in neuronal cells, pntP1 (as well as a modified pntP2 form containing a VP16 transcriptional activation domain, H.S., unpublished observations) can direct ectopic glia differentiation. Interestingly, the com- petence to respond to the pnt signal depends on the segmental position of a given cell [Klaes et al., 19941. In summary, pnt is sufficient and required for several aspects of glia differentiation in the midline as well as in the lateral nerve cord. The determination of glia fate, however, is independent of either gene product. How the glial identity is established in the first place might be learned from studying the transcriptional control of pnt or repo.

CONCLUSIONS To date, we are beginning to understand how glial

differentiation is controlled in the embryonic CNS. But the molecules which eventually perform the different glial cell functions are still unknown. Similarly, the question how glial cell fate is determined in the first place, is largely unsolved and requires further research. Due to the steadily increasing number of cell specific markers and the power of modern genetic analyses, these questions, which are common to a wide range of organisms, can now be efficiently tackled in Drosophila.

ACKNOWLEDGMENTS We thank C. Goodman and S. Harrison for sharing

unpublished data, S. Granderath, T. Kidd, and D. Van Vactor for discussion and comments on the manuscript. The work in the authors lab is supported by the DFG (SFB243 and Hess program) and a Heisenberg-fellow- ship to C.K..

48 KLAMBT ET AL. NOTE ADDED IN PROOF

After the acceptance of this review the gene glial cells missing (gcrn) has been identified. gcm encodes a nuclear protein, which acts as a master gene of lateral glial cell development (Bradley et al., Cell 82, 1013- 1023 and Hosoya et al., Cell 82, 1025-1036).

REFERENCES Anderson MG, Perkins GL, Chittick P, Shrigley RJ, Johnson WA

(1995): Drifter, a Drosophilu POU-domain transcription factor, is required for correct differentiation and migration of tracheal cells and midline glia. Genes Dev 9:123-137.

Auld VJ, Fetter RD, Broadie K, Goodman CS (1995): Gliotactin, a novel transmembrane protein on peripheral glia, is required to form the blood-nerve barrier in Drosophilu. Cell:81:757-767.

Barres BA (1991): New roles for glia. J Neurosci 11:3685-3694. Bastiani MJ, Goodman, CS (1986): Guidance of neuronal growth

cones in the grasshopper embryo. I11 Recognition of specific glial pathways. J Neurosci 6:3542-3551.

Bellen HJ, O’Kane CJ, Wilson C, Grossniklaus U, Kurth-Pearson R, Gehring WJ (1989): P-element-mediated enhancer detection: A ver- satile method to study development in Drosophila. Genes Dev 3:1288-1300.

Bier E, Vassin H, Shephard S, Lee K, McCall K, Barbel S, Ackerman L, Carretto R, Uemara T, Grell EH, J a n LY, Jan YN (1989): Search- ing for pattern and mutations in the DrosophiEa genome with a P-lacZ vector. Genes Dev 3:1273-1287.

Bier E, J a n LY, J a n YN (1990): rhomboid, a gene required for dorso- ventral axis establishment and peripheral nervous system development in Drosophila melanogaster. Genes Dev 4:190-203.

Bossing T, Technau GM (1994): The fate of the CNS midline progenitors in Drosophila as revealed by a new method for single cell labeling. Development 120:1895-1906.

Brunner D, Ducker K, Oellers N, Hafen E, Scholz H, Klambt C (1994): The ETS domain protein PointedP2 is a target of MAP kinase in the Sevenless signaling pathway. Nature 370:386-389.

Buchanan RL, Benzer S (1993): Defective glia in the Drosophilu brain degeneration mutant drop-dead. Neuron 10:839-850.

Campbell G, Goring H, Lin T, Spana E, Andersson S, Doe CQ, Tom- linson A (1994): RK2: A glial-specific homeodomain protein required for embryonic nerve cord condensation and viability in Dro- sophila. Development 120:2957-2966.

Cantera R (1993): Glial cells in adult and developing prothoracic ganglion of the hawk moth Manducu sextu. Cell Tissue Res 272:93-108.

Carlson SD, Saint Marie RL (1990): Structure and function of insect glia. Annu Rev Entomol 35597-621.

Choi K-W, Benzer S (1994): Migration of glia along photoreceptor axons in the developing Drosophila eye. Neuron 12:423-431.

Condron BG, Zinn K (1994): The grasshopper median neuroblast is a multipotent progenitor cell that generates glia and neurons in distinct temporal phases. J Neurosci 145766-5777.

Condron BG, Zinn K (1995): Activation of CAMP-dependent protein kinase triggers a glial to neuronal cell fate switch in an insect neuroblast lineage. Curr Biol 551-61.

Condron BG, Patel NH, Zinn K (1994): Engrailed controls gliaheu- ronal cell fate decisions at the midline of the central nervous system. Neuron 13541-554.

Crews ST, Thomas JB, Goodman CS (1988): The Drosophila single- minded gene encodes a nuclear protein with sequence similarity to the per gene product. Cell 52:143-151.

Dickson B, Hafen E (1994): Genetics of signal transduction in inver- tebrates. Cum Opin Gen Dev 4:64-70.

Doe CQ, Hiromi Y, Gehring WJ, Goodman CS (1988): Expression and function of the segmentation gene fushi tarazu during Drosophila neurogenesis. Science 239:170-175.

Doe CQ, Chu-LaGraff Q, Wright DM, Scott MP (1991): The prosper0 gene specifies cell fates in the Drosophila central nervous system. Cell 65:451-464.

Ebens AJ, Garren H, Cheyette BN, Zipursky SL (1993): The Drosoph- ila anachronism locus: A glycoprotein secreted by glia inhibits neuroblast proliferation. Cell 7415-27.

Edwards JS, Swales LS, Bate M (1993): The differentiation between neuroglia and connective tissue sheath in insect ganglia revisited The neural lamella and perineurial sheath cells are absent in a mesodermless mutant of Drosophila. J Comp Neurol333:301-308.

Escherich K (1902): Zur Entwicklung des Nervensystems der Musci- den, mit besonderer Berucksichtigung des sog Mittelstranges. Zeitschrift fur Wissensch. Zoologie 71:525-549.

Fishell G, Hatten ME (1991): Astrotactin provides a receptor system for CNS neuronal migration. Development 113:755-765.

Freeman M, Klambt C, Goodman CS, Rubin GM (1992): The argos gene encodes a diffusible factor that regulates cell fate decisions in the Drosophilu eye. Cell 69:963-975.

Garriga G, Desai C, Horvitz HR (1993): Cell interactions control the direction of outgrowth, branching and fasciculation of the HSN axons of Coenorhubditis eleguns. Development 117:1071-1087.

Giangrande A (1994): Glia in the fly wing are clonally related to epithelia cells and use the nerve as a pathway for migration. De- velopment 120:523-534.

Giangrande A (1995): Proneural genes influence gliogenesis in Dro- sophila. Development 121:429-438.

Giangrande A, Murray MA, Palka J (1993): Development and orga- nization of glial cells in the peripheral nervous system of Drosoph- ila. Development 11’72395-904.

Gorczyca MG, Phillis RW, Budnik V (1994): The role of tinmun, a mesodermal cell fate gene during the development of the transverse nerve in Drosophila. Development 120:2143-2152.

Halter DA, Urban J, Rickert C, Ner S, It0 K, Travers AA, Technau GM (1995): The homeobox gene rep0 i s required for the differentiation and maintenance of glia function in the embryonic nervous system of Drosophila. Development 121:317-332.

Hamelin M, Zhou Y, Su MW, Scott IM, Culotti J G (1993): Expression of the unc-5 guidance receptor in the touch neurons of Celegans steers their axons dorsally. Nature 364:327-330.

Hatschek B (1877): Beitrage zur Entwicklunsgeschichte der Lepidop- teren. Jen Zeitschrift Natunv BdXI: 115-148.

Hoyle G (1986): Glial cells of an insect ganglion. J Comp Neurol 246:85-103.

It0 K, Urban J, Technau GM (1995): Distribution, classification and development of Drosophila glial cells during late embryogenesis. Roux’s Arch Dev Biol 204:284-307.

Jacobs JR (1993): Perturbed glial scaffold formation precedes axon tract malformation in Drosophilu mutants. J Neurobiol 24:611- 626.

Jacobs JR, Goodman CS (1989a): Embryonic development of axon pathways in the Drosophila CNS: I A glial scaffold appears before the first growth cones. J Neurosci 9:2402-2411.

Jacobs JR, Goodman CS (1989b): Embryonic development of axon pathways in the Drosophila C N S I1 Behavior of pioneer growth cones. J Neurosci 9:2412-2422.

Jacobs JR, Hiromi Y, Patel NH, Goodman CS (1989): Lineage, migration and morphogenesis of longitudinal glia in the Drosophila CNS as revealed by a molecular lineage marker. Neuron 2:1625-1631.

Kennedy TE, Serafini T, de la Torre JR, Tessier-Lavigne M (1994): Netrins are diffusible chemotrophic factors for commissural axons in the embryonic spinal cord. Cell 78:425-435.

Kim SH, Crews ST (1993): Influence of Drosophila ventral epidermal development by the CNS midline cells and spitz class genes. Devel- opment 118:893 -901.

Klaes A, Menne T, Stollewerk A, Scholz H, Klambt C (1994): The ETS transcription factors encoded by the Drosophila gene pointed direct glial cell differentiation in the embryonic CNS. Cell 78:149-160.

Klambt C (1993): The Drosophila gene pointed encodes two ets like proteins, which are involved in the development of the midline glial cells. Development 117:163-176.

Klambt C, Goodman CS (1991): The diversity and pattern of glia during axon pathway formation in the Drosophila embryo. Glia 4205 -213.


Klambt C, Jacobs RJ, Goodman CS (1991): The midline of the Dro- sophila CNS: Model and genetic analysis of cell lineage, cell migration, and development of commissural axon pathways. Cell 64:801- 815.

Kolodkin AL, Pickup AT, Lin DM, Goodman CS, Banejee U (1994): Characterization of Star and its interactions with sevenless and EGF receptor during photoreceptor development in Drosophila. De- velopment 120:1731-1745.

Leptin M, Roth S (1994): Autonomy and non-autonomy in Drosophila mesoderm determination and morphogenesis. Development 120: 853-859.

Martin-Bermudo MD, Carmena A, Jimenez F (1995): Neurogenic genes control gene expression at the transcriptional level in early neurogenesis and in mesectoderm specification. Development 121: 219-224.

Masu Y, Wolff E, Holtman B, Sendtner M, Brern G, Thoenen H (1993): Disruption of the CTNF gene results in motor neuron degeneration. Nature 36527-32,

Mayer U, Nusslein-Volhard C (1988): A group of genes required for pattern formation in the ventral ectoderm of the Drosophila embryo. Genes Dev 21496-1511.

McKanna JA (1993): Primitive glial compartments in the floor plate o f mammalian embryos: Distinct progenitors of adult astrocytes and microglia support the notoplate hypothesis. Perspect Dev Neu- robiol 1245-255.

Menne TV, Klambt C (1994): The formation of commissures in the Drosophila CNS depends on the midline cells and on the Notch gene. Development 120:123-133.

Mitchell KJ, Doyle J, Tear G, Serafini T, Kennedy TE, Mirzyan C, Tessier-Lavigne M, Goodman CS (1994): Genetic analysis of growth cone guidance at the midline in Drosophila: Expression and function of D-netrin. SOC Neurosci Abstr 20:1064.

Nambu JR, Franks RG, Hu S, Crews ST (1990): The single-minded gene of Drosophila is required for the expression of genes important for the development of CNS midline cells. Cell 63:63-75.

Nambu JR, Lewis JO, Wharton KA, Crews ST (1991): The DrosophiZa single-minded gene encodes a helix-loop-helix protein that acts as a master regulator of midline development. Cell 67:1157-1167.

Nelson HB, Laughon A (1993): Drosophila glial architecture and development: Analysis using a collection of new cell specific markers. Roux’s Arch Dev Biol 202:341-354.

OKane C, Gehring WJ (1987): Detection in situ of genomic regulatory elements in Drosophila. Proc Natl Acad Sci USA 84:9123-9127.

ONeill EM, Rebay I, Tjian R, GM Rubin (1994): The activities of two Ets-related transcription factors required for Drosophila eye development are modulated by the RaslMAPK pathway. Cell 78:137- 147.

Poulson DF (1950): Histogenesis, organogenesis, and differentiation in the embryo of Drosophila melanogaster. In Demerec M (ed): “Bi- ology of Drosophila.” New York: John Wiley & Sons, Inc, pp 168- 274.

Prokop A, Technau GM (1994): BrdU incorporation reveals DNA replication in nondividing glial cells in the larval abdominal CNS of Drosophila. RQUX’S Arch Dev Biol 204:54-61.

Rakic P 119711: Neuron-glia relationship during granule cell migration in developing cerebellar cortex: A Golgi and electron microscopic study in Macacus rhesus. J Comp Neurol 141:283-312.

Rakic P (1972): Mode of cell migration in the superficial layers of the fetal monkey neocortex. J Comp Neurobiol 14561-84.

Raz E, Shilo B-Z (1992): Dissection of the faint little ball (flb): Phe- notype: Determination of the development of the Drosophila central nervous system by early interactions in the ectoderm. Development 114:113-123.

Reynold ML, Woolf CJ (1993): Reciprocal Schwann cell-axon interactions. Curr Op in Neurobiol 3586-693.

Rothberg JM, Hartley DA, Walther Z, Artavanis-Tsakonas S (1988): slit: An EGF-homologous locus in Drosophila melanogaster involved in the develoDment of the embrvonic central nervous svstem.

Rothberg JM, Jacobs JR, Goodman CS, Artavanis-Tsakonas S (1990): slit: An extracellular protein necessary for development of midline glia and commissural axon pathways contains both EGF and LRR domains. Genes Dev 4:2169-2187.

Rutledge BJ, Zhang K, Bier E, J a n YN, Perrimon N (1992): The Drosophila gene spitz encodes a putative EGF-like growth factor involved in dorsal-ventral axis formation and neurogenesis. Genes Dev 6:1503-1517.

Schoenwolf GC, Smith J L (1990): Mechanisms of neurulation: Tradi- tional view point and recent advances. Development 109:243- 270.

Seeger M, Tear G, Ferres-Marca D, Goodman CS (1993): Mutations affecting growth cone guidance in Drosophila: Genes necessary for guidance towards or away from the midline. Neuron 10:409-426.

Serafini T, Kennedy TE, Galko MJ, Mirzayan C, Jessel TM, Tessier- Lavigne M (1994): The netrins define a family of axon outgrowth- promoting proteins homologus to Celegans UNC-6. Cell 78:409- 424.

Silver J, Lorenz SE, Wahlstein D, Coughlin J (1982): Axonal guidance during the development of the great cerebral commissures: Descrip- tive and experimental studies in vivo on the role of preformed glial pathways. J Comp Neurol210:10-29.

Singer M, Nordlander RH, Edgar M (1979): Axonal guidance during embryogenesis and regeneration in the spinal cord of the newt: The blue print hypothesis of neuronal patterning. J Comp Neurol 185: 1-22.

Sonnenfeld MJ, Jacobs JR (1994): Mesectodermal cell fate analysis in Drosophila midline mutants. Mechanisms of Development 46:3-13.

Sonnenfeld MJ, Jacobs JR (1995): Apoptosis of the midline glia during Drosophila embryogenesis: A correlation with axon contact. Devel- opment 121:569-578.

Stollewerk A, Klambt C, Cantera R (1995): Electron microscopic analysis of midline glial cell development during embryogenesis and larval development using P-galactosidase expression as endogenous cell marker. Microsc Res Tech (in press).

Sturtevant MA, Roark M, Bier E (1993): The Drosophila rhomboid gene mediates the localized formation of wing veins and interacts genetically with components of the EGF-R signaling pathway. Genes Dev 7:961-973.

Thomas JB, Crews ST, Goodman CS (1988): Molecular genetics of the single-minded locus: A gene involved in the development of the Drosophila nervous system. Cell 52133-141.

Udolph G, Prokop A, Bossing T, Technau GM (1993): A common pre- cursor for glia and neurons in the embryonic CNS of Drosophila gives rise to segment specific lineage variants. Development 118: 765-775.

Vincent JP, Kassis JA, O’Farrell PH (1990): A synthetic homeodomain binding site acts as a cell type specific, promoter specific enhancer in Drosophila embryos. EMBO d 925734578,

Wemmer T, Klambt C (1995): A genetic analysis of the Drosophila genes bulge, argos and soba; three closely linked, interacting genes. Genetics (in press).

Wharton KA, Crews ST (1993): CNS midline enhancers of the Dro- sophila slit and Toll genes. Mechanisms of Development 40:141- 154.

Wigglesworth VB (1959): The histology of the nervous system of an insect Rhodnius prolixus. I1 The central ganglia. Q J Microsc Sci 100:299-314.

Winberg ML, Perez SE, $teller H (1992): Generation and early differentiation of glial cells in the first optic ganglion of Drosophila rnelanogaster. Development 115:903-91 1.

Xiong WC, Montell C (1995): Defective glia induce neuronal apoptosis in the repo visual system of Drosophila. Neuron 14581-590.

Xiong WC, Okano H, Pate1 NH, Blendy JA, Montell C (1994): rep0 encodes a glia-specific homeo domain protein required in the Dro- sophila nervous system. Genes Dev 8:981-994.

Zak NB, Wides RJ, Schejter ED, Raz E, Shilo B-Z (1990): Localization of the DERlflb protein in embryos: Implications on the faint little

Cell 55:1047-1059. ball lethal phenotype. Development 109:865-874.

development and function of embryonic central nervous system glial cells indrosophila

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