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First published online 3 March 2004
doi: 10.1242/dev.01057

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Cell movements controlled by the Notch signalling cascade during foregut development in Drosophila

Bernhard Fuss^*, Frank Josten^*, Maritta Feix and Michael Hoch

Universität Bonn, Institut für Molekulare Physiologie und Entwicklungsbiologie, Abteilung für Molekulare Entwicklungsbiologie, Poppelsdorfer Schloss, D-53115 Bonn, Germany

View larger version (114K): [in a new window]   Fig. 1. Cell movement during proventriculus development. Developing proventriculi of stage 12 (A,E,I), stage 14 (B,F,J), stage 15 (C,G,K) and stage 17 (D,H,L) wild-type embryos. (A-D) Anti-Fkh (red)/anti-Dve (green) marking ectodermal and endodermal cells, respectively. A constriction separates the ectodermal and endodermal part of the keyhole (arrow in B, see also F). (E-H) anti-Armadillo staining. Note the concentration of Arm towards the apical side of the cells (arrow in E,F). (I-L) Anti-Arm(red)/anti-MHC (blue) immunostaining. The mesoderm-free region that lacks MHC expression is marked by arrows in I. (M-Q) Schematic representation of the different stages of proventriculus development and the cell movements resulting in the invagination of the ectodermal keyhole cells. Ectodermal cells in orange, endodermal cells in green and mesodermal cells in blue.

View larger version (134K): [in a new window]   Fig. 2. Expression of members of the Notch signalling pathway during proventriculus development. (A-D) Dl expression in the ectodermal cells of the keyhole monitored by Anti-Dl (red)/anti-Dve (green) immunostaining at stage 12 (A), stage 15 (B), stage 16 (C) and late stage 17 (D). Delta localises to the ectodermal keyhole domain which moves into the endodermal cell layer. Arrows (C,D) mark the downregulation of Dl expression in anterior (ac) and posterior (pc) boundary cells. (E-L) Dynamic Notch receptor expression visualised in a single channel visualisation (E-H) and in an anti-Notch (red)/anti-Dve (green) immunostaining (I-L). (E,I) Stage 13; (F,J) stage 14; (G,K) stage 15; (H,L) late stage 17 wild-type embryos. The Notch receptor is upregulated in ac and pc. (M-P) Relative localisation Delta (green) and Notch signalling activity [Gbe-Su(H)m8-lacZ; red] during proventriculus development. The epithelial gut tube is surrounded by broken lines; ac and pc are highlighted by arrows. Notch signalling activity is restricted to the ac and pc, which are adjacent to the Delta expression domain.

View larger version (77K): [in a new window]   Fig. 3. Notch signalling is required for cell movements during proventriculus morphogenesis. Anti-Fkh(red)/anti-Dve (green) immunostaining of Dl (A-C), Notch (D-F), fng (G-I) and Su(H) (J-L) loss-of-function mutants at stage 12 (A,D,G,J), stage 15 (B,E,H,K) and stage 17 (C,F,I,L) of proventriculus development. Specification of the early proventriculus primordium is not affected in any of the mutants (compare with wild type, Fig. 1A), whereas cell movements leading to the keyhole structure at stage 15 (compare with wild type, Fig. 1C) and to the cardia structure at stage 17 (compare with wild type, Fig. 1D) do not take place, leading to block of invagination in mutants of the Notch signalling cassette. (M) Ectopic 14-3fkh-Gal4 mediated expression of the Notch ligand Dl causes a Notch-like phenotype, i.e. loss of invagination of ectodermal cells, as shown by anti-Dl (red)/anti-Dve (green) double staining. (N) Ectopic hsGal4 mediated expression of the Notch extracellular domain (NECD) also abrogates infolding of ectodermal cells at late stages of proventriculus development, visualised by anti-Dl(red)/anti-Dve(green) double staining. (O) Anti-Fkh (red)/anti-Dve (green) double staining showing that ectopic activation of the Notch signalling pathway causes ectopic cell movements (arrow). However, we do not observe changes in endodermal or ectodermal cell fate in these embryos.

View larger version (119K): [in a new window]   Fig. 4. Shot expression during proventriculus development. (A-H) Anti-Shot (red)/anti-Dve (green) antibody stainings of wild-type embryos of stage 12 (A,E), 14 (B,F) and 17 (C,G, tangential section; D,H, sagittal section). (A-D) Single channel visualisation of Shot expression. Shot localises to the apical side of the ectodermal (ec) keyhole domain (lower arrow in A) and to the apical and basal sides of the neighbouring endodermal cells that are covered by visceral mesoderm (upper arrow in A). During invagination, Shot protein is upregulated on the apical side of the posterior boundary cells (pc in C,D). Shot expression is reduced in the ac (D,H). (I) Anti-Shot (red)/anti-Arm (green)/anti-MHC (blue) immunostaining at stage 17 visualising uniform expression of Arm throughout the proventriculus epithelium and locally restricted elevation of Shot in the pc. (J-L) Anti-Fkh(red)/anti-Dve (green) immunostaining of shot mutants at stage 12 (J), stage 15 (K) and stage 17 (L) revealing the failure of ectodermal cells to invaginate and a collapse of the proventricular endoderm. (M-O) Phalloidin stainings visualising the actin cytoskeleton of stage 17 wild-type embryos (M,N) and a cdc42 mutant embryo (O). In wild type, actin filaments accumulate on the apical side of pc (M) whereas lower levels of actin filaments are seen in the ac that move inward (N, arrow). (O) In cdc42 mutants, cell movements leading to the keyhole structure are not initiated, the endodermal proventriculus epithelium is collapsed and ectodermal cells fail to invaginate. (P) Anti-Shot (red)/anti-Arm (green)/anti-MHC(blue) triple staining of a late stage 17 embryo in which a dominant-negative form of the GTPase Cdc42 was expressed in the posterior boundary cells. Note the failure of invagination.

View larger version (64K): [in a new window]   Fig. 5. shot is a Notch target gene and required for Notch signalling. (A,B) Anti-Shot (red)/anti-Dve (green) immunostaining of stage 17 wild-type (A) and Notch (B) mutant embryos. Upregulation of Shot expression in the posterior boundary cells (pc) does not occur in Notch mutants (arrow). (C) Shot mRNA expression in a wild-type embryo of stage 17 and in an embryo in which the Notch pathway has been ectopically activated (D). Note the ectopic activation of shot transcription as compared with wild type. (E,F) Anti-NotchECD immunostaining of the proventriculus primordium of stage 14 wild-type (E) and shot mutant embryos (F). Arrows in E and F indicate the keyhole. Notch receptor expression is strongly reduced in the ectodermal keyhole (ky) domain in shot mutants. (G,H) Anti-ß-Gal(red)/anti-Fas3 (green) double staining of late stage 14 Gbe-Su(H)m8-lacZ embryos in a wild-type (G) and a shot mutant embryo (H); note the loss of reporter gene expression in pc. (I,J) Anti-ßGal(red)/anti-Dve (green) double staining of stage 17 Gbe-Su(H)m8-lacZ embryos in wild-type (I) and shot mutant background (J). The activity of the Notch signalling pathway is strongly reduced in the posterior boundary cells (pc).

View larger version (52K): [in a new window]   Fig. 6. Model of Notch signalling controlling cell movement in the proventriculus. (A) Schematic illustration of the proventriculus primordium in late stage 13 (left) and stage 15 (right), highlighting the expression domains of proventriculus regulators and the cell movement events. Mesoderm in blue; ectoderm in orange; endoderm in green; ac, anterior boundary cells; pc, posterior boundary cells. Arrows highlight the inward movement of the ac. Note that the number of cells in the mesoderm-free region is about 10. For a better overview of the localisation of gene activities in the proventriculus primordium, only maximum gene activities are highlighted in the cells. The expression domains of regulators of proventricular development are shown in B. For further information, see text.