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First published online 19 November 2003
doi: 10.1242/dev.00882

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Echinoid facilitates Notch pathway signalling during Drosophila neurogenesis through functional interaction with Delta

Emma L. Rawlins^*, Bridget Lovegrove and Andrew P. Jarman^,

The Wellcome Trust Centre for Cell Biology, Institute of Cell and Molecular Biology, University of Edinburgh, King's Buildings, Edinburgh EH9 3JR, UK

View larger version (121K): [in a new window]   Fig. 1. echinoid mutants display additional es organs and SOPs. (A,B) Scanning electron microscopy of the adult thorax. (A) Wild type. (B) edlH23/edl(2)k01102 showing additional macrochaetae around each wild-type macrochaete location. (C) Light micrograph of sca-Gal4; UAS-ed thorax. Arrows indicate the positions where macrochaetae should be present. An arrowhead indicates two macrochaetae that have arisen from the same PNC in place of one. (D-J) Confocal microscopy for immunohistochemical detection of (D-G) Sens, and (H-J) Ac (green) and Sens (red) in third larval instar wing discs. (D) Wild type. (E) edlH23/edl(2)k01102. (F,G) Higher magnification views of scutellar region in (D,E). Note that two SOPs are present at each position (one of the SOPs in F has divided and appears as two adjacent cells). (H) Wild type. (I) edlH23/edl(2)k01102 showing no difference in Ac level or pattern compared with wild type. (J) sca-Gal4; UAS-ed wing disc at puparium formation. Arrows indicate proneural clusters that have persisted much later than normal in the absence of SOP specification.

View larger version (119K): [in a new window]   Fig. 2. echinoid interaction with Notch and sca. (A) Light micrograph of N55e11/+; ed4.12/ed4.12 adult thorax. The arrows indicate the positions that macrochaetae should occupy. (B) Immunohistochemical detection of Elav (neurons) in the N55e11/+; ed4.12/ed4.12 pupal thorax. Note that the neurons are arranged in groups rather than singly as expected for wild type. (C,D) Immunohistochemical detection of Sca (green) and Sens (red) in third larval instar wing discs. (C) Wild type. (D) edLH23/edl(2)k01102. Sca expression is unaffected in the ed mutant background.

View larger version (124K): [in a new window]   Fig. 3. Echinoid subcellular localisation and colocalisation with Notch and Delta. Third instar larval discs examined by confocal microscopy. (A) Ed protein in wild-type wing disc. (B) sca-Gal4/UAS-ed-GFP. Detection of Ed-GFP (green) and Ed (red). (C) sca-Gal4/UAS-fng-DXDmut-Myc. Ed (red) and Golgi represented by Myc (green). Arrows indicate some Ed protein that is in the Golgi. Arrowheads indicate some of the more numerous Ed-only vesicles. (D) Immunohistochemical detection of Sca (green) and Ed (red), showing lack of colocalisation. (A-D) Single confocal sections. (E) Immunohistological detection of Ed (green), HRS (red) and NECD (blue). Confocal xzy projection. Arrows indicate vesicles containing all three proteins. (E') Green channel (Ed). (E'') Red channel (NECD). (E''') Blue channel (HRS). (F,G) Ed expression in confocal projections. (F) Wild type. (G) hook1 mutant, showing fewer Ed-positive vesicles. (H,I) Immnuohistochemical detection of Dl (green) and Ed (red). Single confocal sections of wild-type discs. (H) Wing disc. Note that the majority of the Ed colocalises with Dl; some of the Ed- and Dl-positive vesicles are marked by arrows. Dl-only vesicles are marked by arrowheads. (I) Eye disc. Ed does not colocalise very strongly with Dl. (J) Immunohistochemical detection of Ed-GFP (green), NECD (red) and Dl (blue). Confocal xzy projection. Arrows indicate vesicles containing all three proteins. (J') Green channel (Ed-GFP). (J'') Red channel (NECD). (J''') Blue channel (Dl).

View larger version (140K): [in a new window]   Fig. 4. Overexpression of Echinoid affects Dl levels but not N. Single confocal sections of dpp-Gal4/UAS-ed wing discs. (A,B) Immunohistochemical detection of Ed (green) and Dl (red). (A) Apical cell surface. (A') Red channel (Dl). (B) Confocal section taken just below the apical cell surface. (B') Red channel (Dl). (C) Immunohistochemical detection of Ed (green) and NECD (red). (C') Red channel (NECD). The levels of Dl fluorescence are decreased both at the cell membrane and in intracellular vesicles while the levels of N are unaffected.

View larger version (74K): [in a new window]   Fig. 5. Echinoid is associated with the cis-endocytosis of Dl in S2 cells. Immunohistochemical detection and confocal microscopy of N, Dl, Ed-Myc and Ed-FLAG in transiently transfected S2 cells. (A) Ed-Myc-expressing cell (green) contacting an Ed-FLAG-expressing cell (red). Ed is visible at the region of contact between the cells and in cis-endocytic vesicles (arrows). (B) Single N- (NICD, green) and Dl (red)-expressing cell. (C) N- (NICD, green), Dl- (red) and Ed-FLAG (blue)-expressing cell contacting an Ed-Myc-expressing cell. Arrows indicate cis-endocytic vesicles containing all three proteins. The anti-FLAG antibody also shows some non-specific staining of cell nuclei. (D) Single Dl- (red) and Ed-FLAG (blue)-expressing cell. Dl and Ed are frequently located in the same vesicle (arrows). (E) N- (NECD, green) and Ed-Myc-expressing cell (not detected) contacting a Dl- (red) and Ed-FLAG (blue)-expressing cell. Ed is cis-endocytosed with Dl as N is trans-endocytosed (arrows). Trans-endocytosis of N or Dl alone is not associated with Ed (arrowheads). (F) N (NECD, green) and Ed-FLAG (blue)-expressing cell contacting a Dl- (red) and Ed-Myc (not visible)-expressing cell. Ed is not trans-endocytosed with N into the signalling cell. Trans-endocytosis of N or Dl alone is not associated with Ed (arrowheads). (G) Cartoon summary of S2 cell experiments. (G') In isolated S2 cells Ed is associated with the cisendocytosis of Dl/N together (1) and Dl alone (2), but not with N alone (3). (G'') In contacting S2 cells Ed is associated with the cisendocytosis of Dl (1), which is necessary for trans-endocytosis of NECD during N activation (Parks et al., 2000). It is not associated with other trans-endocytosis events seen in S2 cells that are unrelated to N activation (2, 3).

View larger version (164K): [in a new window]   Fig. 6. Genetic mosaic analysis of Echinoid. (A,B) Mosaic analysis of edLH23 in third larval instar imaginal discs examined by confocal microscopy. (A) Immunohistological detection of Ase (green) and ß-galactosidase (red) in an edLH23 genetic clone in a wing disc. The edLH23 homozygous region is marked by the absence of ß-galactosidase and the clone border has been marked by a white line. The arrows indicate cell-autonomous specification of SOPs. The arrowhead labels a pair of adjacent SOPs; one mutant, one wild type. (A') The green channel (Ase). (B) Immunohistological detection of Ed (red) in an edLH23 mosaic leg disc. The edLH23 homozygous region is marked by the absence of nlsGFP (green), the remainder of the disc is either wild type or heterozygous for edLH23. The mutant cells express very little Ed; the wild-type cells at the clone border show a large drop in the amount of Ed on the region of membrane that touches the mutant cells of clone. (B') The red channel (Ed) from B.

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