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doi: 10.1242/10.1242/dev.00415

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Neuroglian activates Echinoid to antagonize the Drosophila EGF receptor signaling pathway

Rafique Islam^1,*, Shu-Yi Wei^2,*, Wei-Hsin Chiu², Michael Hortsch^1, and Jui-Chou Hsu^2,

¹ Department of Cell and Developmental Biology, University of Michigan, Medical School, Ann Arbor, MI 48109-0616, USA
² Institute of Molecular Medicine, Department of Life Science, National Tsing Hua University, Hsinchu, Taiwan 30043, Republic of China

View larger version (140K): [in a new window]   Fig. 1. Overexpression of Ed and Nrg results in loss of photoreceptor and cone cells. Scanning electron micrographs (A-D) of adult eyes, and midpupal eye imaginal discs stained for the photoreceptor marker ELAV (red, E-H) and cone cell marker Cut (green, I-L). (A,E,I) Wild type; (B,F,J) GMR-GAL4/UAS-ed; (C,G,K) GMR-GAL4/UAS-nrg180; (D,H,L) GMR-GAL4/UAS-ed/UAS-nrg180. (I-L) The upper focal planes of E-H, respectively. There are seven photoreceptors (E, R8 is out of the plane of focus) and four overlying cone cells (I) in wild-type imaginal discs. Overexpression of UAS-ed in eye causes a mild rough eye (B), ommatida with six or fewer photoreceptor neurons (arrowhead in F), and ommatidia with three or fewer cone cells (J). The arrows in F and J indicate the same ommatidium that contains seven photoreceptor (F) and three cone cells (J). Overexpression of nrg180 (C,G,K) alone causes no phenotype at 25°C. However, co-expression of both ed and nrg180 results in a more severe rough eye phenotype (D), as manifested by reduced number of ommatidia, varying size of ommatidia and decreased number of bristles. In addition, a much higher percentage of ommatidia contain fewer photoreceptor (arrows in H) and cone cells (arrows in L).

View larger version (158K): [in a new window]   Fig. 2. Ed is colocalized with Nrg. Third instar larval eye discs were double-labeled with anti-Ed antibodies (green) and anti-Nrg monoclonal antibody 1B7 (red). Both Ed and Nrg are co-expressed in the basal undifferentiated cells (A-C) and in developing photoreceptor clusters (D-F). Scale bar: 10 µm.

View larger version (12K): [in a new window]   Fig. 3. Western blot analysis of Ed and Nrg protein expressed by transfected S2 cell lines. Shown is a western blot analysis of induced, native (lane 1) and transfected S2 cells. Each lane contains the total protein from 2.5x105 cells. Lane 2 represents cells that were transfected with a construct encoding the neuronal isoform of Nrg, whereas lane 3 contains cells that were transfected with a HA epitope-tagged version of Drosophila Ed. Blot A was incubated with the anti-Nrg monoclonal antibody BP-104 and blot B with the HA.11 monoclonal antibody, respectively.

View larger version (93K): [in a new window]   Fig. 4. Ed protein induces homophilic cell aggregation of S2 cells. Shown are the results of a S2 cell mixing experiment, in which unlabeled and DiI-labeled S2 cells were mixed at a ratio of 1: 1 and co-aggregated. (A,B) Two views of areas with labeled and unlabeled S2 cells, which are both expressing Ed protein. The left part of each panel depicts the view using Nomarski optics, whereas the same area is shown in the right part of the panel using epifluorescence with a rhodamine filter set. In C,D, unlabeled Ed-expressing S2 cells were co-aggregated with DiI-labeled, native S2 cells. Scale bar: 70 µm.

View larger version (101K): [in a new window]   Fig. 5. Ed and Nrg protein are heterophilic binding partners, when expressed on separate populations of transfected S2 cells. Shown are representative areas of four combinations of S2 cell mixtures, which have been co-aggregated. The left sides of all panels show areas of S2 cells using Nomarski optics, with the same areas shown on the right side using epifluorescence, thereby visualizing the DiI-labeled cells. For all experiments, DiI-labeled cells and unlabeled cells were mixed at a ration of 1: 1 and subsequently aggregated on a shaking platform. (A) A mixture of DiI-labeled, Ed-expressing cells with unlabeled, Nrg-expressing S2 cells; (B) DiI-labeled, native S2 cells with unlabeled, Nrg-expressing cells; (C) DiI-labeled, Ed-expressing cells with unlabeled, Fasciclin 1-expressing cells; (D) DiI-labeled, native S2 cells with unlabeled, Fasciclin 1-expressing S2 cells. The inserts in C depict a small aggregate of S2 cells, which consists entirely of DiI-labeled and therefore Ed-expressing cells. Scale bar: 70 µm.

View larger version (14K): [in a new window]   Fig. 6. Co-immunoprecipitation of Ed and Neuroglian protein from mixed S2 cell aggregates. For this co-immunoprecipitation experiments S2 cells expressing Ed were mixed and co-aggregated with either S2 cells expressing NrgGPI (lanes 1 and 3) or native S2 cells (lane 2). Soluble proteins were immunoprecipitated using the anti-Nrg monoclonal antibody 1B7 (lanes 1 and 2) or a control monoclonal antibody (lane 3). The western blot shown in this figure was analyzed for the presence of Ed protein using the HA.11 monoclonal antibody.

View larger version (59K): [in a new window]   Fig. 7. Ed acts in the signal-receiving cells. (A) Nrg derivatives: Nrg180 is the full-length neuron-specific protein isoform and contains the Nrg extracellular, the Nrg transmembrane (TM) and the Nrg180 intracellular domain. NrgGPI contains only the Nrg extracellular domain and is anchored to the plasma membrane by the Fasciclin 1 GPI moiety (Hortsch et al., 1995). (B) Ed derivatives: Ed contains the complete extracellular and TM Ed domains, followed by the entire 315 amino acid intracellular Ed domain. EdDintra lacks the intracellular Ed domain. EdC50 contains the Ed TM domain and the last C-terminal 50 amino acid of Ed. (C-E) Scanning electron micrographs of adult eyes. (F-K) Eye discs stained for the photoreceptor marker ELAV (red, F-H) and cone cell marker Cut (green, I-K). (C,F,I) GMR-GAL4/UAS-nrgGPI; (D,G,J) GMR-GAL4/UAS-ed/UAS-nrgGPI; (E,H,K) GMR-GAL4/UAS-edC50. (I-K) Upper focal planes of (F-H), respectively. Overexpression of nrgGPI (C,F,I) alone causes no apparent phenotype at 25°C. However, co-expression of both ed and nrgGPI results in a severe rough eye phenotype (D). A similar phenotype is induced by the overexpression of edC50 (arrows in H,K). Arrows in G,J indicate ommatidia contain fewer photoreceptor and cone cells, respectively.

View larger version (19K): [in a new window]   Fig. 8. Model of Echinoid-Neuroglian homophilic and heterophilic interactions. Echinoid and Neuroglian are both homophilic CAMs. They also trans-interact with each other via a heterophilic mechanism. Whether they are also able to cis-interact in the same plasma membrane is currently unknown. Most significantly, L1-type proteins are cell-autonomous, positive regulators of EGFR signaling, whereas Ed negatively influences EGFR function via its cytoplasmic domain. By interacting with Ed that is expressed on neighboring cells Neuroglian can act as an autonomous activator and as a non-autonomous inhibitor of EGFR signaling.