Netrins guide migration of distinct glial cells in the Drosophila embryo

Abstract

SUMMARYDevelopment of the nervous system and establishment of complex neuronal networks require the concerted activity of different signalling events and guidance cues, which include Netrins and their receptors. In Drosophila, two Netrins are expressed during embryogenesis by cells of the ventral midline and serve as attractant or repellent cues for navigating axons. We asked whether glial cells, which are also motile, are guided by similar cues to axons, and analysed the influence of Netrins and their receptors on glial cell migration during embryonic development. We show that in Netrin mutants, two distinct populations of glial cells are affected: longitudinal glia (LG) fail to migrate medially in the early stages of neurogenesis, whereas distinct embryonic peripheral glia (ePG) do not properly migrate laterally into the periphery. We further show that early Netrin-dependent guidance of LG requires expression of the receptor Frazzled (Fra) already in the precursor cell. At these early stages, Netrins are not yet expressed by cells of the ventral midline and we provide evidence for a novel Netrin source within the neurogenic region that includes neuroblasts. Later in development, most ePG transiently express uncoordinated 5 (unc5) during their migratory phase. In unc5 mutants, however, two of these cells in particular exhibit defective migration and stall in, or close to, the central nervous system. Both phenotypes are reversible in cell-specific rescue experiments, indicating that Netrin-mediated signalling via Fra (in LG) or Unc5 (in ePG) is a cellautonomous effect.

DrosophilaGlial Glutamate Transporter Eaat1 Is Regulated by Fringe-Mediated Notch Signaling and Is Essential for Larval Locomotion

“…Monoclonal antibodies obtained from the Developmental Studies Hybridoma Bank included the following: mouse anti-Prospero (dilution 1:25); mouse anti-Fas2 (1:10); mouse anti-Even-skipped (Eve) (dilution 1:10); and mouse anti-Repo (1:50). Other antibodies used were as follows: rabbit anti-GFP (1:1000, Invitrogen); rabbit anti-␤-gal (1:1000, MP Biomedicals); mouse anti-glutamine synthetase 2 (Gs2) (1:100, Millipore Bioscience Research Reagents); rabbit anti-cleaved caspase-3 (1:100, Cell Signaling Technology); rabbit anti-VGlut (1:400) (Mahr and Aberle, 2006); and rabbit anti-Nazgul (Naz) (1:500) (von Hilchen et al, 2010). Secondary antibodies used were as follows: Alexa Fluor 488-conjugated goat anti-rabbit and goat anti-mouse (1:300, Invitrogen); Alexa Fluor 647-conjugated goat anti-mouse (1:300, Invitrogen); and Rhodamine Red-X-conjugated goat anti-rabbit and goat anti-mouse (1:300, Jackson ImmunoResearch).…”

Neuroblast pattern and identity in the Drosophila tail region and role of doublesex in the survival of sex-specific precursors

“…The following primary antibodies were used: mouse anti-Abdominal B (1:20) (Celniker et al, 1989), rat anti-Elav (1:2000), mouse anti-Invected (1:2) (Patel et al, 1989), mouse anti-Sex lethal (1:10) (Bopp et al, 1991) and mouse anti-Wrapper (1:20) (Noordermeer et al, 1998) (all from Developmental Studies Hybridoma Bank); chicken anti-Beta-Gal (1:1000) (Abcam); rabbit anti-Castor (1:500) (Kambadur et al, 1998) (provided by Ward Odenwald); guinea pig anti-Dbx (1:1500) (Lacin et al, 2009) (provided by James Skeath); rabbit anti-Deadpan (1:100) (Bier et al, 1992) (provided by Harald Vaessin); rat anti-Doublesex (1:100) (Sanders and Arbeitman, 2008) (provided by Michelle Arbeitman); rabbit anti-Eagle (1:500) (Dittrich et al, 1997); rat anti-Empty spiracles (1:1000) (Walldorf and Gehring, 1992) and rabbit anti-Eyeless (1:1000) (Kammermeier et al, 2001) (provided by Uwe Walldorf); rabbit anti-Engrailed (1:100) (Santa Cruz Biotechnology); rabbit anti-Even skipped (1:1000) (Frasch et al, 1987) (provided by Manfred Frasch); mouse anti-GFP (1:250) (Roche); rabbit anti-GFP (1:500) (Torrey Pines Biolabs); rat anti-Gooseberry distal (1:2) and rat anti-Gooseberry proximal (1:2) (Zhang et al, 1994) (provided by Robert Holmgren); guinea pig anti-Hunchback (1:1000) (Mettler et al, 2006) (provided by Joachim Urban); mouse anti-Ladybird early (1:2) (Jagla et al, 1997) (provided by Krzysztof Jagla); rabbit anti-mCherry (1:500) (Bio Vision); rabbit anti-Miranda (1:100) (Betschinger et al, 2006) (provided by Juergen Knoblich); rabbit anti-Msh (1:500) (provided by Matthew Scott, Stanford University, USA); rabbit anti-Nazgul (1:400) (von Hilchen et al, 2010) and guinea pig anti-Reversed polarity (1:10,000) (provided by Benjamin Altenhein); guinea pig anti-Orthodenticle (1:500) (Xie et al, 2007) (provided by Tiffany Cook); rabbit anti-RFP (1:500) (MBL); guinea pig anti-Runt (1:500) (Kosman et al, 1998) For in situ hybridisation, we used a digoxygenin-labelled ind RNA-probe (provided by Matthew Scott). It was synthesised as described previously (Urbach and Technau, 2003b).…”

Spinster Controls Dpp Signaling during Glial Migration in theDrosophilaEye

“…Glial migration is extensively studied in Drosophila. During embryonic PNS development, glial cells born in the CNS migrate outward along the segmental nerves guided by diffusible signals and differential adhesion (Sepp et al, 2000;von Hilchen et al, 2008von Hilchen et al, , 2010. During wing formation, peripherally born glia follow sensory axons as they travel toward the CNS (Aigouy et al, 2004(Aigouy et al, , 2008.…”