The Midline Glia of Drosophila: a molecular genetic model for the developmental functions of Glia
Introduction
The nervous system provides an ideal playground for developmental biologists seeking to unravel the machinery of cell determination, differentiation and cell to cell communication. In part, this is because nervous tissue is composed of a bewildering diversity of cell types. Each of these cells must establish their own identity, morphology, connectivity and physiology to become a functional component of the nervous system. One would predict a plethora of lineage specific gene expression events during nervous system development. Indeed, 50% of spatially regulated genomic enhancers can regulate expression within the developing nervous system (Bellen et al., 1989, Bellen et al., 1990). In nervous tissue one may identify each neuron as unique and examine gene expression and function from that perspective.
Glia can be identified as individuals too! Glial biologists remind their neuron-centric colleagues that the mature mammalian brain contains many more glia than neurons. From a developmental perspective, the eminence of glia is also revealed by their earlier emergence and differentiation. For example, the radial glia of the vertebrate cerebrum establish the morphological template upon which cortical neuronal networks are built (Bentivoglio and Mazzarello, 1999, Rakic, 1978). After neurons reach their appropriate location, having migrated along a glial substrate, they extend axonal processes, whose guidance is instructed by attractive or repellent contact cues on glial cell surfaces, or in the extracellular matrix that glia synthesize (Auld, 1999, Goodman, 1996, Silver, 1994). Glial cells subsequently provide trophic, nutritive, insulating and housekeeping services to neurons and contribute to intercellular signaling (Vernadakis, 1988, Vernadakis, 1996).
We are beginning to uncover the molecular basis of glial functions in development. In vertebrate models, much of what we know comes from expression studies in situ and manipulative studies in tissue culture (Ahmad et al., 1999, Trotter, 1993). Random or targeted genetic perturbation of glial function, amenable in genetic models of development has made less headway in the vertebrate nervous system. The roles of glial expressed genes in guiding neuron migration and axon ensheathment has been revealed in the mouse by mutations in the jimpy and myelin associated glycoprotein genes (Bartsch, 1996, Vela et al., 1998).
The mutant approach to understanding glial function has a significant advantage: function is first assessed by mutant phenotype. Each mutation is randomly generated by chemical or transposon mutagenesis. There are no a priori assumptions about the molecular identity of the gene product that may bias our selection of candidate genes. The mutant approach therefore has the best chance of uncovering novel genes and new functions for known genes. Among the genetic models of nervous system development, Drosophila has proven to be the most fertile in uncovering gene functions in glia during development (Giangrande, 1996, Klambt et al., 1996). Without regard to the evolutionary distance between insects and vertebrates, most of these functions have been determined for vertebrate glia also, often employing orthologous gene products in conserved functional pathways.
Drosophila glial biologists cannot boast that glia predominate numerically or in volume in their model nervous system. Nevertheless, the early emergence of glia in development reflects their contributions to CNS morphogenesis. Drosophila glia have been classified into many families based on morphology, reflecting their roles in guidance, ensheathment, scavenger functions and forming the blood brain barrier (Carlson and Saint Marie, 1990, Hoyle, 1986). The diversity of glia is also revealed by enhancer trap studies. Genomic enhancers regulating gene expression in subsets of cells may be detected by reporter genes (such as GFP) placed randomly by transposon insertion (Bellen et al., 1990, Yeh et al., 1995). Enhancer trap studies reveal the diversity of genes expressed by glia, and serve as tools to follow lineage and development. Integration of morphological, lineage and gene expression data enables a classification of glial types based upon functional criteria (Hartenstein et al., 1998, Ito et al., 1995, Jacobs et al., 1989) reviewed by Klambt et al. (1996). The functional diversity of Drosophila glia is beyond the scope of this review. Instead, I shall focus upon the glial cell about which we have learned more about gene expression and function than any other CNS cell type in Drosophila, the Midline Glia (MG).
Pattern in developing tissue depends upon the establishment of boundaries which communicate positional information to neighbouring cells. Boundaries naturally emerge in appendages or in serially repeated structures like segments. The universal boundary of bilaterally organised metazoans is the midline. It communicates medial to lateral positional information. In deuterostomes and protostomes, the midline is established by the interface between mesoderm and ectoderm during gastrulation. In vertebrates, the midline structure is the notochord, and an ectodermal derivative it determines, the floorplate of the neural tube (reviewed by Placzek et al., 1990). In Drosophila, midline cells are the mesectoderm, which demarcate the ectoderm and mesoderm during gastrulation (Poulson and Demerec, 1950, Thomas et al., 1988, Kosman et al., 1991). The Drosophila mesectoderm and the vertebrate notochord and floorplate are common elements of a conserved body plan that is similar between vertebrates and invertebrates (Arendt and Nubler-Jung, 1996, Arendt and Nubler-Jung, 1999). This conserved element generates the cells of the midline of the nervous system, which are the first CNS cells to be determined in embryogenesis (Schoenwolf and Smith, 1990, Schoenwolf and Smith, 1990, Crews et al., 1988, Thomas et al., 1988). Midline cells subsequently communicate medial to lateral positional information to the CNS (Lee et al., 1999, Skeath, 1998, Placzek et al., 1991, Yamada et al., 1991, i Altaba, 1998), the ectoderm (Schweitzer et al., 1995a, Schweitzer et al., 1995b, Kim and Crews, 1993) and the mesoderm (Lewis and Crews, 1994, Luer et al., 1997). In the nervous system, “positional information” is reflected developmentally in two manners. First, position contributes to cell identity. If one disturbs the cell to cell signaling that communicates position relative to a boundary, then cells acquire the wrong identity, often leading to duplication or deletion of specific cell types (Lee et al., 1999, Skeath, 1998). Second, position contributes to guidance and the establishment of connectivity. Growing axons find their targets of innervation by detecting a series of intermediate targets along the way (Auld, 1999, Goodman, 1996). Altering the positional identity of those targets will interfere with correct “pathfinding”.
In vertebrates, the floorplate acts as a gatekeeper, permitting only those axons which must establish contralateral connections to cross the midline (Bernhardt et al., 1992a, Bernhardt et al., 1992b, Colamarino and Tessier-Lavigne, 1995a, Colamarino and Tessier-Lavigne, 1995b). The MG perform an equivalent role in the Drosophila embryo (see below). Loss of the floorplate and notochord in the Danforth short tail and HNF-3β mutants results in a loss of midline axon projections and disrupts longitudinal tracts as well (Bovolenta and Dodd, 1991, Weinstein et al., 1994). Boundary functions are similarly provided in the brain, although not all midline cells of the brain arise from the vertebrate floorplate or fly mesectoderm. Cells of the optic chiasm (Reese et al., 1994) or the corpus callosum, for instance, provide a substrate that selectively allows contralateral axon extension (Richards et al., 1997, Wang et al., 1996). The roof plate of the tectum provides a boundary for axon and dendrite extension (Snow et al., 1990). These “boundary cells” express molecules such as tenascin and chondroitin sulfate proteoglycan, that inhibit axon outgrowth (Silver, 1994, Steindler, 1993). Removal of these cells allows misprojection of axons and dendrites across the midline (Wu et al., 1998).
Many midline cell types are transient cells with a morphogenetic function. In other words, the cells exist primarily to provide positional information, and to guide and mould the morphology of other cells. Once this function is complete, the cells are superfluous, and are removed. In vertebrates, cells at the midline are removed by apoptosis when their contribution to boundary establishment is no longer required (Knyihar-Csillik et al., 1995, Hankin et al., 1988, Laywell and Steindler, 1991). Similarly, the MG of Drosophila undergo two rounds of programmed cell death, the first after establishment of commissural connections in the embryo, and the second during pupation, when the adult nervous system midline structure is established (Sonnenfeld and Jacobs, 1995a, Sonnenfeld and Jacobs, 1995b, Perz, 1994, Stollewerk et al., 1996).
A single cell type, the MG of the Drosophila CNS, is the fly analogue of the floorplate, and contributes to all analogous boundary and guidance functions. A full understanding of how the MG arise and how they behave reveals most of the developmental mechanisms of midline boundary function. The MG received attention in early studies of insect nervous system morphogenesis because these cells arise early in development, and they are among the largest cells of the developing nerve cord. Midline glia were first noted as ensheathing cells in the grasshopper (Wheeler, 1893) and the housefly (Springer and Rutschky, 1969). Ultrastructural studies of the Drosophila embryo implicated an early role in morphogenesis of the midline and establishment of the commissural tracts as shown in Fig. 1 (Jacobs and Goodman, 1989). Subsequently the MG differentiate as ensheathing glia of the embryonic and larval nervous system (Jacobs and Goodman, 1989, Stollewerk et al., 1996). Morphological and genetic analysis of the MG has further revealed that these cells perform many functions attributed to vertebrate oligodendroglia, including ensheathment (Jacobs and Goodman, 1989), axon guidance (Battye et al., 1999, Kidd et al., 1999, Mitchell et al., 1996), phagocytosis (Sonnenfeld and Jacobs, 1995a, Sonnenfeld and Jacobs, 1995b), and establishment of the blood brain barrier (Baumgartner et al., 1996) and midline cytoarchitecture (Sonnenfeld and Jacobs, 1994).
Molecular and genetic studies of MG function have progressed rapidly in the last decade, in part, because the prominent cells are easy to identify and are easy targets for detection of gene expression or directed transgene expression (employing the GAL4 expression system; Brand and Perrimon, 1993). Perturbed MG differentiation results in distinct CNS morphology phenotypes, easily selected from a mutagenesis (Hummel et al., 1999a, Hummel et al., 1999b). For these reasons, we have learned more about gene expression in the MG than any other embryonic CNS cell type. Expression or function of nearly 50 genes, excluding ubiquitously expressed ones, have been established in the MG lineage (Table 1). Below I will examine what we have learned about the origin and differentiation of this unique cell type, and then examine what genetic studies have revealed about the many functions of the MG.
Section snippets
Function and determination of the mesectoderm
Early function of the MG cannot be isolated from early functions of mesectodermal cells (MECs) because many signals generated by the MG are generated first by some or all of the mesectoderm. These functions include medial to lateral signals that influence cell identity (including the Drosophila EGF pathway) and midline guidance (including the Netrins and Slit).
As a protostome, the Drosophila embryo gastrulates from the ventral surface of the blastoderm. Ventral blastoderm cells will become the
MG lineage specific gene expression
For decades, vertebrate glial biologists have relied upon simple differentiation markers, like GFAP, an astrocyte specific intermediate filament, to identify glia. Insect neurobiologists have lacked such a tool, and have often been asked to defend to their vertebrate colleagues how they know with certainty that any cell under study is actually a glial cell. This issue has been put to rest by the identification of “master regulatory genes” of glial determination and differentiation. Expression
MG function in axon guidance
The majority of interneurons with projecting axons make contralateral connections. These axons cross the midline once, never making ipsilateral connections after decussation. During development, these axons must be guided towards and across the midline at an appropriate location, and subsequently instructed not to re-approach the midline. Neurons making only ipsilateral connections must also be instructed not to approach the midline. How do growth cones know where the midline is, and how do
Extracellular matrix
Another developmental function attributed to glia is the production of extracellular matrix (Bunge, 1993, Pearlman and Sheppard, 1996). Many molecules inserted by the MG into the peri-axonal ECM are functionally associated with facilitating or guiding axon extension. These include Slit, Laminin and Syndecan (Fessler and Fessler, 1989, Montell and Goodman, 1989, Rothberg et al., 1990, Spring et al., 1994). Other secreted proteins like Tiggrin and Masquerade, although not produced by the MG, also
Glial-axonal trophic interactions
Signaling between glia and the axons they ensheath appears to be bi-directional. In vertebrates, neuronal-glial adhesion (such as L1) and exchange of trophic factors (such as Neuregulin and Insulin-like Growth factor-1) mediate survival and maintain differentiation of both cell types (Gassmann and Lemke, 1997, Haney et al., 1999, Syroid et al., 1999). In Drosophila, MG survival depends upon axon ensheathment. Survival of the MG requires Wrapper, which appears to relay contact signals from
Prospects
The MG are required for multiple essential roles in the development and function of the CNS. Genetic studies have revealed a number of MG specific activities. As a result we probably know more about the MG than we know about any other Drosophila CNS cell type. The horizon continues to expand for many aspects of MG biology. Lineage tracing studies and analysis of segment polarity mutants will likely resolve the mechanism of MG determination. The function of Egfr signaling is now clear, but we do
Acknowledgements
Thanks to M. Sonnenfeld, C. Stemerdink, R. Dong, B. Lanoue, M. Perz, R. Battye and A. Stevens for data used in the figures. Fig. 7 was made with the help of A. Brand. C. Nurse and A. Campos provided helpful comments on the manuscript. This research has been supported by the Natural Sciences and Engineering Research Council of Canada, the Multiple Sclerosis Society of Canada, and the Medical Research Council.
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