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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.

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