The glycosyltransferase Fringe promotes Delta-Notch signaling between neurons and glia, and is required for subtype-specific glial gene expression

Graham B. Thomas 2 Donald J. van Meyel ) 0 1 3 0 Department of Neurology and Neurosurgery, McGill University 1 Centre for Research in Neuroscience 2 Graduate Program in Neurological Sciences 3 McGill University Health Centre Research Institute , 1650 Cedar Avenue, Montreal, QC, H3G 1A4 , Canada The development, organization and function of central nervous systems depend on interactions between neurons and glial cells. However, the molecular signals that regulate neuron-glial communication remain elusive. In the ventral nerve cord of Drosophila, the close association of the longitudinal glia (LG) with the neuropil provides an excellent opportunity to identify and characterize neuron-glial signals in vivo. We have found that the activity and restricted expression of the glycosyltransferase Fringe (Fng) renders a subset of LG sensitive to activation of signaling through the Notch (N) receptor. This is the first report showing that modulation of N signaling by Fng is important for central nervous system development in any organism. In each hemisegment of the nerve cord the transcription factor Prospero (Pros) is selectively expressed in the six most anterior LG. Pros expression is specifically reduced in fng mutants, and is blocked by antagonism of the N pathway. The N ligand Delta (Dl), which is expressed by a subset of neurons, cooperates with Fng for N signaling in the anterior LG, leading to subtype-specific expression of Pros. Furthermore, ectopic Pros expression in posterior LG can be triggered by Fng, and by Dl derived from neurons but not glia. This effect can be mimicked by direct activation of the N pathway within glia. Our genetic studies suggest that Fng sensitizes N on glia to axon-derived Dl and that enhanced neuron-glial communication through this ligand-receptor pair is required for the proper molecular diversity of glial cell subtypes in the developing nervous system. INTRODUCTION Interactions between neurons and glial cells are vital for the development, function, plasticity and maintenance of nervous systems (Freeman and Doherty, 2006). During nervous system development in Drosophila, glial cells are important for neuron survival and for the guidance, pruning and ensheathment of axons; in return, neurons provide trophic support for glia, stimulate glial proliferation and guide glial migrations (Booth et al., 2000; Chotard and Salecker, 2004; Hidalgo and Griffiths, 2004; Klambt et al., 2001). Although progress has been made in understanding cellular aspects of neuron-glial interactions, the molecular signals that regulate communication between neurons and glial cells remain largely undetermined. In the ventral nerve cord (VNC) of Drosophila, several glial classes have been defined based on their morphology, position, function and additional embryologic and molecular characteristics (Ito et al., 1995). The longitudinal glia (LG) lie dorsal to the longitudinal axon tracts of the VNC (Jacobs et al., 1989). These axon tracts, called connectives, lie within a dense neuropil enwrapped by plasma membrane from LG. The close association of LG with the neuropil provides an excellent system to identify and characterize neuron-glial signals in vivo. LG are derived from a glioblast, which gives rise to 10-12 glial progeny only (Griffiths and Hidalgo, 2004; Jacobs et al., 1989; Schmidt et al., 1997). Proliferation within this lineage is controlled in part by Epidermal growth factor receptor (EGFR) signaling in response to an axon-derived signal, Vein (Hidalgo et al., 2001). Mitosis occurs in LG precursors that express the transcription factor Prospero (Pros) at high levels (Griffiths and Hidalgo, 2004). Pros is thought to promote cell division through EGFR-dependent activation of the MAPK pathway. Of the 10-12 LG at late stages of embryogenesis, only six continue to express Pros. Subclasses of glia such as LG are highly specialized in both form and function, and probably arise from intrinsic genetic programs as well as extrinsic cues experienced in part through contact with neurons. All glia in Drosophila, except midline glia, are intrinsically specified by a regulatory cascade of transcription factors including Glial cells missing (Gcm), Tramtrack, PointedP1 and Reversed polarity (Repo), which act in concert to promote glial-specific gene expression (Jones, 2005). Targets of this cascade encode the regulator of G-protein signaling Locomotion defects (Loco) and the transcription factor Retained (Retn) (Granderath et al., 1999; Shandala et al., 2002; Yuasa et al., 2003). However, Loco and Retn are expressed in only restricted subsets of glia, including LG, as are a number of other genes, including the Fibroblast growth factor receptor Heartless (Htl), the transcription factor Distal-less and Pros. The individual or combined activities of these and other factors are likely to endow LG with their specialized morphological and functional properties. Indeed, genetic mutants for retn exhibit defects of LG position and reduced Loco and Pros expression, while mutants of loco or htl each have defects in LG membrane morphology (Granderath et al., 1999; Shandala et al., 2003; Shishido et al., 1997). The restriction of gene expression to specific subsets of glia suggests there may be context-dependent, locally derived regulators that direct aspects of glial differentiation, including extrinsic molecular signals provided by axons. The expression of Pros in only six LG, and its absence in the remainder of the LG with which they share a common lineage, provided an opportunity to identify and characterize local molecular signals that mediate the differentiation of glial subtypes. We have found that expression of fringe (fng) is restricted to a small population of cells in the VNC that include a subset of LG. Fng is a -1,3-N-acetyl-glucosaminyl (GlcNac) transferase that catalyzes the addition of GlcNac to O-linked fucose monosaccharides on specific EGF repeats of the extracellular domain of the Notch (N) receptor (Blair, 2000; Haltiwanger and Stanley, 2002). N is a single-pass transmembrane receptor that has two known ligands in Drosophila, Serrate (Ser) and Delta (Dl). Fng modulates signaling through N by reducing the sensitivity of N for Ser and increasing its sensitivity for Dl (Bruckner et al., 2000; de Celis and Bray, 2000; Fleming et al., 1997; Hicks et al., 2000; Moloney et al., 2000; Okajima et al., 2003; Panin et al., 1997). Only some of the many developmental events controlled by N also involve Fng. In Drosophila, Fng is an important determinant of boundary formation in the wing, eye and leg imaginal discs, and for specifying polar cell fates during oogenesis (Cho and Choi, 1998; de Celis et al., 1998; Dominguez and de Celis, 1998; Grammont and Irvine, 2001; Irvine and Wieschaus, 1994; Klein and Arias, 1998; Rauskolb et al., 1999; Rauskolb and Irvine, 1999). To date, modulation of N by Fng has not been implicated in central nervous system (CNS) development in any organ (...truncated)