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The Drosophila gap junction channel gene innexin 2 controls foregut development in response to Wingless signalling

Reinhard Bauer^*, Corinna Lehmann^*, Bernhard Fuss, Franka Eckardt and Michael Hoch

Institut für Zoophysiologie der Universität Bonn, Abt. für Entwicklungsbiologie, Poppelsdorfer Schloss, 53115 Bonn, Germany
^* These authors contributed equally to this work

View larger version (84K): [in a new window]   Fig. 1. Reporter gene expression pattern of the kropfP188 line and location of the P insertions in the innexin 2 transcription unit. (A-F) ß-Gal expression monitored by anti-ß-Gal antibody staining of whole-mount embryos. (A) Stage 10 embryo, showing uniform expression in the germ band (arrow). With the beginning of germ band retraction (B), the ubiquitous expression pattern resolves into localised expression in the ectodermal fore- and hindgut, the endodermal anterior and posterior midgut, and the excretory Malpighian tubule primordia. During germ band retraction, reporter gene expression is maintained in these tissues (C-E) and in stage 17 embryos (E,F), expression occurs predominantly in the endodermal parts of the proventriculus and in the small intestine in the hindgut. (G) The localisation of the P-element insertions is indicated by inverted triangles. The genomic organisation of the transcript is shown below (Stebbings et al., 2000). (H) In situ hybridisation analysis of wild type (H) and of a kropf mutant embryo (I) using antisense probes for innexin 2 (inx2) and orthopedia (otp; internal staining control). Note that the inx2 pattern is abolished in kropf mutants, whereas otp is still expressed. amg, anterior midgut; fg, foregut; hg, hindgut; MT, Malpighian tubules; pmg, posterior midgut; pv, proventriculus; si, small intestine.

View larger version (96K): [in a new window]   Fig. 4. Apical localisation of innexin 2 mRNA. In situ hybridisation using digoxigenin (A-D) or fluorescein-labelled RNA innexin 2 antisense riboprobes (E-G). (A) innexin 2 transcripts are detected in the cytoplasm of the nurse cells (nc) and the oocyte (oc) during oogenesis. Note the localisation of the mRNA in the cortical region of the oocyte and the border cells (arrows). (B) Blastoderm embryo. Transcripts are ubiquitously distributed. (C) Germ band extension stage. mRNA expression occurs in the invaginating stomodeum (st), the anterior midgut primordium (amg) that abuts the involuted foregut tube, in the proctodeum (p) and in segment precursors (Stebbings et al., 2000). (D) During keyhole formation, innexin 2 expression is found in the ectodermal (ec) and endodermal (en) tissue regions of the proventriculus primordium (arrow marks the boundary). Note that the innexin 2 mRNA expression pattern corresponds to the reporter gene expression pattern of the P lines (compare with Fig. 1). (E-G) Double immunostaining of a proventriculus of a stage 16 embryo (marked by the stippled line) using a fluorescein-labelled RNA innexin 2 antisense riboprobe (E; red) and anti-Discs lost antibody (F; green), which marks the apical (ap, shown by arrows) sides of epithelial cells (Bhat et al., 1999). innexin 2 mRNA is localised to the apical sides of the proventricular cells (merge in G).

View larger version (89K): [in a new window]   Fig. 2. kropf mutant larvae display a feeding defect. (A) Wild-type first instar larva fed with red dyed food (feeding assay). The food is predominantly found in the midgut. The multi-layered proventriculus is transparent (arrow). (B) In homozygous kropf mutant larvae, the food is stuck at the proventriculus (arrow). (C) Magnification of the proventriculus of the wild-type larva shown in A. Note that the ectodermal cells (ec) have migrated into the endodermal pouch (en, arrows). (D) Magnification of the proventriculus of the kropf mutant larva shown in B. Note that the ectodermal cells (ec) have failed to move into the endodermal pouch (en; arrows). As a result, the red food is stuck in the oesophagus causing a feeding defect.

View larger version (124K): [in a new window]   Fig. 3. Keyhole formation is disrupted in kropf P mutants. (A,B) Wild-type embryos stained with anti-Wingless antibody. (C,D) Magnifications of the keyhole region shown in A,B. (A) Stage 14 embryo. Note the striped Wingless expression domain prior to keyhole formation (arrow; magnification in C). (B) Stage 15 embryo. Note that the Wingless expression domain is split (arrow; magnification in D). (E,F) Anti-Wingless antibody staining of kropf mutant embryos. (E) Stage 14 embryo. Note that the striped Wingless expression domain is not affect in kropf mutants (magnification in G) when compared with wild type. (F) Stage 15 embryo. Note that the Wingless expression domain fails to split (arrow; magnification in H). (I,J) Confocal images of anti-Fkh (green)/anti-Dve (red) staining of stage 17 wild-type (I) and kropf mutant embryos (J). Although in wild type, the ectodermal cells (ec) have invaginated into the endodermal proventriculus pouch, the ectodermal cells are stuck on top of the endoderm in kropf P mutants (compare with the larval phenotype in Fig. 2D).

View larger version (77K): [in a new window]   Fig. 5. innexin 2 transcription is induced by Wingless signalling. The ß-Gal expression of the kropfP16 line in stage 15 embryos of wild-type (A,C), homozygous wingless mutants (B) and in embryos in which ß-Gal has been ectopically expressed by using the twi-Gal4 driver and the UAS-wingless effector lines (see text). Note that reporter gene expression is abolished in the proventriculus region in wingless mutants (compare regions marked by arrows in A,B), whereas it is dramatically expanded in the anterior and posterior region in the Wingless overexpression experiment (compare region marked by arrows in C,D). (E) RT-PCR experiments in Drosophila tissue culture S2 cells (Materials and Methods). Schneider S2 cells were either transfected with an expression vector for Armadillo, the ß-catenin homologue that has been shown to serve as the transducer of the Wingless signalling cascade (pIBArm) or with the vector alone (pIB). innexin 2 mRNA levels were monitored by performing RT-PCR analysis (actin mRNA levels served as an internal control). Note that innexin 2 mRNA levels are increased approximately five times in response to Armadillo when compared with the control reaction. (F) The regionalisation of developing gut tube. The foregut (fg) and the hindgut (hg) are of ectodermal (brown) and the midgut (mg) of endodermal origin (blue). At the ectoderm/endoderm boundaries of the gut, signalling centres arise that control the development of the proventriculus (pv) and the small intestine (si) in the anterior region and in the posterior regions, respectively. A working model suggests that Wingless (green) activates innexin 2 transcription (red) via the signal transducer Armadillo (right) in the boundary regions of the gut. Note, however, that the arrows do not imply direct molecular interactions (see Discussion). This induction may be required for enhanced gap junctional communication during the morphogenetic processes in both gut parts.

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