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First published online March 6, 2009
doi: 10.1242/10.1242/dev.030254

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Neurexin IV and Wrapper interactions mediate Drosophila midline glial migration and axonal ensheathment

¹ Department of Biochemistry and Biophysics, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
² Department of Cell and Molecular Physiology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
³ Curriculum in Neurobiology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
⁴ Department of Biology, The University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.

View larger version (133K): [in this window] [in a new window]   Fig. 1. MG migration and commissural axon ensheathment and subdivision. (A-F') Composite confocal images of sim-Gal4 UAS-tau-GFP Drosophila embryos in sagittal views. (A,A') Single segment and (B-F') high-magnification views focused primarily on MG and axon commissures. Anterior is left and dorsal (internal) is up. Developmental stage and orientations are shown in the upper right corner. Sag, sagittal; ven, ventral. White asterisks, anterior midline glia (AMG); orange asterisks, posterior midline glia (PMG); 1, MP1 neurons; a, anterior commissure (AC); p, posterior commissure (PC). MP1 neurons were identified by their characteristic shape, position and axonal trajectories. (A,A') At stage 12/3, the AMG and PMG moved internally, contacting the single commissure (arrow). Only two AMG and three PMG are shown in this focal plane. The position of midline neurons is shown for reference (bracket). (B,B') At stage 12/0, the AMG began a posterior migration along the dorsal (white arrow) and ventral (yellow arrow) aspects of the commissure. (C,C') During stage 12/0, the AMG extended a cytoplasmic process between the AC and PC (arrow). (D,D') Following the process, an AMG (red asterisk) migrated between the AC and PC. (E,E') A single, dorsally located AMG (magenta asterisk) began to migrate over the PC during stage 15. A small gap (arrow) was still present between AMG and PMG. Inset shows GFP staining in the boxed region, showing that the AMG extended projections (arrowheads) into the AC. (F,F') During stage 17, the more posterior of the dorsal AMG (magenta asterisk) sent out a thin process (arrow) that surrounded the PC. Glial membranes continued to project into the commissures (arrowheads in inset). (G) Schematic summary of MG migration. The schematic depicts an idealized view; actual segments vary in MG number and position. Sagittal views. Axon commissures, pink; circles indicate midline cell nuclei surrounded by cytoplasmic Tau-GFP, green; MP1s, blue (1); AMG, gray (A); PMG, gray (P); midline neurons, green. Dashed lines indicate nuclei of cells undergoing apoptosis. Individual cell borders are not shown.

View larger version (125K): [in this window] [in a new window]   Fig. 2. Nrx-IV is required for MG migration and commissural axon ensheathment and subdivision. Wild-type (A) and Nrx-IV mutant sim-Gal4 UAS-tau-GFP (B-K) Drosophila embryos. (A) In wild type, AMG were between the AC and PC (arrow). Inset illustrates that AMG projections were present within the boundaries of the commissure (dashed lines). (B) In Nrx-IV mutants, the commissures were wider and poorly separated; AMG were not present between the commissures (arrow) and did not extend projections (inset). (C) Nrx-IV mutant AMG and PMG migrated dorsally toward the commissure (arrow). (D) AMG contacted the commissure (thin arrow) along its anterior side (white arrow), and the PMG contacted the commissure along its posterior side (yellow arrow). (E,E') During stage 13, an AMG (red asterisk) migrated posteriorly across the AC, failed to ensheath the AC, and instead moved directly toward the PC. The PMG and MP1 neurons were positioned normally. (F) By stage 15, the AC was often surrounded by AMG, but AMG membranes were not closely associated with the AC along its posterior side (arrow). (G) At stage 13, AMG were often not in contact with the MP1 neurons (arrow indicates the gap) and the AC was not completely surrounded by AMG processes. (H) At stage 17 in Nrx-IV mutants, AMG were dissociated from the MP1 neurons (anti-Lim3); arrow indicates the gap. (I) AMG surrounded the AC, but not the PC, and AMG failed to send projections into the AC (inset) or PC. (J) Failure of AMG to surround either commissure. (K) AMG were completely absent; two commissures were apparent, but they were diffuse.

View larger version (137K): [in this window] [in a new window]   Fig. 3. Nrx-IV localizes to MG-neuron interfaces. (A) In situ hybridization for Nrx-IV RNA in wild-type Drosophila embryos showed relatively uniform low levels of expression throughout the CNS. Brackets, lateral CNS; horizontal bar, midline. (B,B') Anti-Nrx-IV showed high protein concentrations (arrows) at the midline, and much lower levels in lateral CNS neurons (B', anti-Elav). (C,C') sim-Gal4 UAS-tau-GFP embryos were stained with anti-Nrx-IV and anti-GFP. Nrx-IV was mainly localized: (1) inside the commissures (between the dashed lines), and (2) along the borders of AMG (white arrow) and PMG (yellow arrow) with neurons. Staining was not observed at MG-MG boundaries (arrowhead). (D,D') At stage 13, Nrx-IV was localized to the contact points (arrow) between AMG and the BP102+ unseparated commissure. (E,E') During stage 15, Nrx-IV was localized at the boundary of the BP102+ AC with AMG (yellow arrow) and on AMG processes infiltrating the commissure (white arrow). (F) Nrx-IV was highly localized (arrow) to the membranes of Elav+ lateral CNS neurons, where they contacted AMG. (G) At stage 14, Nrx-IV was highly concentrated at the long interface (arrow) between AMG and the Lim3+ MP1 neurons. The Lim3+ cells above the MG are non-midline cells. (H,H') During stage 13, Nrx-IV-GFP localized to the membranes of Elav+ neurons (arrow) and was enriched at the boundaries where neurons (white arrowhead) and commissural axons (yellow arrowhead) were adjacent to Wrapper+ AMG. (I,I') At stage 15, Nrx-IV-GFP was localized to axons in the commissures (a, p) and longitudinal connectives (white arrowheads), and to the contact points (yellow arrowhead) between neurons and Wrapper+ AMG. (J,J') At stage 14, in Nrx-IV- elav-Gal4 UAS-Nrx-IV, Nrx-IV was concentrated at the points of contact between neurons and both AMG (white arrow) and PMG (yellow arrow). (K) In stage 15 wild-type embryos, the AC (BP102) was ensheathed by AMG (Wrapper) and an AMG (arrow) was inserted between the AC and PC. (L) In Nrx-IV-, AMG (Wrapper) failed to surround the AC (BP102) or migrate between the commissures; arrow indicates the position of the missing AMG. (M) Similar to wild type, Nrx-IV- elav-Gal4 UAS-Nrx-IV AMG (Wrapper) ensheathed the AC (BP102) and moved between the AC and PC (arrow).

View larger version (113K): [in this window] [in a new window]   Fig. 4. wrapper is required for AMG migration and commissure ensheathment and subdivision. (A) wrapper in situ hybridization on wild-type Drosophila embryos showing expression in AMG (arrow). (B-E') Wild-type and (F-I) wrapper- embryos containing sim-Gal4 UAS-tau-GFP. (B,B') Wrapper protein was observed throughout the AMG, including the membrane (arrow), which lies just outside of cytoplasmic Tau-GFP staining. Wrapper is also present at MG-MG boundaries (between the arrowheads). (C) During AMG migration, Wrapper localized to AMG membranes surrounding the AC (arrow) and at the boundary with the MP1 neurons (between the arrowheads). (D,D') Wrapper was present but not concentrated at the interfaces with Elav+ neurons. (E,E') Wrapper localized along the MG projections (arrow) within the BP102+ AC and PC. (F) At stage 15, AMG failed to completely ensheath the AC, and migrated toward the PC. A gap was present between the AMG and MP1 neurons (arrow). (G) At stage 17, AMG loosely surrounded the AC, but not the PC. MG projections were absent from the AC (inset). (H) AMG failed to ensheath either commissure. (I) Lim3 showed the position of one MP1 neuron and its dissociation from the AMG.

View larger version (140K): [in this window] [in a new window]   Fig. 5. Nrx-IV membrane localization is dependent on wrapper. (A-D') sim-Gal4 UAS-tau-GFP (A,A',C,C') and wrapper- sim-Gal4 UAS-tau-GFP (B,B',D,D') Drosophila embryos. (A,A') Nrx-IV was highly concentrated at the boundary of AMG and lateral CNS neurons (yellow arrow), and weakly present along the neuronal membrane not touching the AMG (white arrow). (B,B') In wrapper mutants, Nrx-IV was not localized to the boundary with AMG (yellow arrow), but was instead uniformly localized around the entire membrane of lateral CNS neurons. (C,C') Nrx-IV was concentrated at the boundaries between AMG and the BP102+ AC (arrow). (D,D') In wrapper mutants, mislocalized Nrx-IV was present at high levels within the BP102+ AC and PC, rather than at the boundaries with AMG. Nrx-IV was not observed in AMG. (E,E') In en-Gal4 UAS-GFP-lacZ.nls, Nrx-IV was not concentrated at the contact points between β-Gal+ and β-Gal- cells (arrow). Wrapper was undetectable because it is not endogenously expressed in these cells. (F,F') In en-Gal4 UAS-wrapper embryos, Nrx-IV was highly concentrated at the boundaries of Wrapper+ En+ cells at sites of contact with Wrapper- En- cells (arrow). Nrx-IV did not accumulate at the borders of adjacent Wrapper+ cells (arrowhead). Inset shows that Wrapper was either localized to the membrane (white arrowhead) or present in puncta (yellow arrowhead).

View larger version (52K): [in this window] [in a new window]   Fig. 6. Nrx-IV and Wrapper bind and mediate cell adhesion. (A) arm-Gal4 UAS-wrapper embryonic lysates were probed on immunoblots (IB) with anti-Nrx-IV after anti-Wrapper immunoprecipitation (IP), and with anti-Wrapper after anti-Nrx-IV immunoprecipitation. This showed that Wrapper and Nrx-IV exist in a protein complex. In negative control experiments on wild-type lysates, anti-Nrx-IV did not immunoprecipitate Neurotactin (Nrt). (B) Anti-Nrx-IV immunoblot showing the presence of Nrx-IV in cells treated with control pBluescript RNAi (pBS), and absence of Nrx-IV in cells treated with two different Nrx-IV dsRNAs (Nrx1, Nrx2). Anti-Tubulin controlled for input protein levels. (C) Summary of RNAi experiments. y-axis shows the percentage of Wrapper+ cells found in aggregates with 2 cells, averaged between two experiments. Error bars show s.d. (D,E) Drosophila S2 cells were transiently transfected with (D) Nrx-IV alone and (E) wrapper alone. (F) Nrx-IV- and wrapper-transfected cells were mixed together. (G-I) S2 cells were transiently transfected with wrapper and immunostained for Wrapper and Nrx-IV. Wrapper- cells are outlined (dotted lines). Scale bar: 5 µm

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