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Mechanisms of Gurken-dependent pipe regulation and the robustness of dorsoventral patterning in Drosophila

Francesca Peri, Martin Technau and Siegfried Roth*

Institut für Entwicklungsbiologie, Universität zu Köln, Gyrhofstr. 17, D-50923 Köln, Germany



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  		Fig. 1. Expression of pipe mRNA and a pipe reporter construct in wild-type ovaries. All egg chambers are oriented with the anterior towards the left and dorsal towards the top. (A-E) pipe mRNA distribution. (F-K) pipe-lacZ expression visualized using anti-ß-galactosidase antibodies. (A,F) During early stage 9, pipe is expressed in two domains, a weak anterior domain and a strong posterior-ventral domain. (B,G,C,H) The two domains of expression approach each other during stage 9 and (D,I) fuse, forming a solid ventral stripe at early stage 10. (E) pipe mRNA disappears during stage 10 starting from the posterior pole. (J) Stage 12. (K) Stage 14.

 


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  		Fig. 2. Raf loss-of-function and {lambda}top flip-out clones affect pipe expression in a cell-autonomous manner. (A-I) Stage 10 egg chambers (anterior towards the left and dorsal towards the top). Anterior (A) and posterior (D) clones of mutant cells lacking Raf are marked by the absence of GFP (green) expression. (B,E) pipe expression is visualized using a pipe-lacZ construct (red). (C,F) The merged pictures show that pipe is de-repressed in Raf mutant clones (lack of green) located at the dorsal side. De-repression occurs in a cell-autonomous manner and it is not influenced by the position of the clone along the AP axis. (G-I) {lambda}top flip-out clones. (G) {lambda}top-expressing cells are marked by GFP expression (green). (H) pipe expression is visualized using a pipe-lacZ construct (red). (C) The merged pictures show that {lambda}top expression leads to cell-autonomous repression of pipe.

 


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  		Fig. 3. pipe expression is not affected by dof and rho loss-of-function clones, but by ectopic expression of rho. (A-I) Stage 10 egg chambers (anterior towards the left and dorsal towards the top). (A) A large clone of mutant cells missing dof is marked by the absence of GFP expression (green). (B) pipe expression is visualized using a pipe-lacZ construct (red). (C) The merged pictures show that pipe expression is not affected by loss of dof activity. (D) A large clone lacking rho function is marked by the absence of GFP. (E) pipe expression is visualized using the pipe-lacZ construct. (F) The merged pictures show that pipe expression is not affected by loss of rho activity. (G) rho flip-out clones marked by GFP expression. (H) pipe expression is visualized using the pipe-lacZ construct. (I) The merged pictures show that ectopic rho expression (green) leads to cell-non-autonomous repression of pipe (red).

 


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  		Fig. 4. fringe mutant clones do not affect pipe expression. Large anterior (A) and posterior (D) clones that lack fng are marked by the absence of GFP expression (green). (B,E) pipe expression is visualized using the pipe-lacZ construct (red). (C,F) The merged pictures show that fng clones do not affect pipe expression.

 


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  		Fig. 5. The effects of large dec-marked Raf clones on the larval cuticle. (A1,B1) Darkfield micrographs of the lateral surface of eggs focusing on the outer eggshell (chorion). The chorion derived from dec follicle cell clones appears to be more transparent. Anterior is towards the left and dorsal to the top. (A2,B2,A3,B3) Camera lucida drawings of the left and right sides of the same eggs. The clones are marked in pink. (A4,B4) Camera lucida drawings of the larval cuticle present inside the eggs. da, dorsal appendage; de, dorsal epidermis; fk, filzkörper; hs, head skeleton; vd, ventral denticles. (A) Egg carrying a large ventral clone that contains a larva with normal cuticle. (B) Egg carrying a large dorsolateral clone. Deletions of head and thorax structures and the expansion of ventral denticle rows of the first abdominal segment (arrow) might be caused by local ventralization.

 


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  		Fig. 6. The effects of dec-marked Raf clones of variable sizes on the larval cuticle. Darkfield micrographs of the (A1,B1) dorsal or (C1) lateral surface of eggs focusing on the outer egg shell (chorion). The chorion derived from dec follicle cell clones appears to be more transparent. Anterior is towards the left. (A2,B2,C2) Camera lucida drawings of the pictures shown in A1,B1,C1. The dec clones are marked in pink. (A3,B3,C3) Nomarski micrographs showing the larval cuticle inside the eggs. (A4,B4,C4) Camera lucida drawings of the larval cuticles. (A5,B5,C5) Phase-contrast micrographs showing magnified views of the regions of the cuticle boxed in A3,B3,C3. (A) Egg carrying multiple dorsal dec Raf clones that contains a larva with normal cuticle. At the dorsal side facing the clones the cuticle harbours dorsal hairs (A5). (B) Egg carrying a large dorsal dec Raf clone, which comprises about 40 follicle cells and spans up to 10 follicle cells in the mediolateral dimension. The larva that developed inside this egg has a normal cuticle. At the dorsal side facing the clone, the cuticle harbours dorsal hairs (B5). (C) Egg carrying a large lateral dec Raf clone that comprises about 40 follicle cells. The larva that developed inside this egg shows cuticular defects in the head and thorax region which might be caused by a local ventralization. At the posterior pole, the presence of normally sized ventral denticle belts (vd) and extended filzkörper (fk, B5) demonstrates normal DV patterning.

 


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  		Fig. 7. Effects of Raf clones on twist expression. twi and ftz protein distribution in gastrulating embryos. All embryos are shown from the ventral side. Anterior is towards the left. (A) Embryo showing normal twi expression. The twi-expressing cells invaginate along the ventral midline (ventral furrow). The embryo is derived from an egg collection in which the eggs carried several Raf clones, ranging from 2-16 cells in size. (B-E) Embryos derived from eggs carrying clones with an average size of 30-60 cells. (B,C) The ventral furrow is displaced to lateral positions as it runs along the AP axis. (D,E) Local expansions of the twi domain are accompanied by a bifurcation of the ventral furrow. In some embryos, similar effects were also seen at more posterior positions.

 

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