Fig. 6. Ectopic tracheal expression of Grh inhibits branch extension. (A) mAb2A12 (lumenal) labelling of a btl-GAL4/UAS-grh embryo at stage 16 reveals that primary branch extension is impeded. No secondary branches are detectable and the individual tracheal segments fail to connect (compare to wild type in Fig. 1A). (B,C) Dorsal view of stage 16 wild-type (B) and btl-Gal4/UAS-grh (C) embryos labelled for DSRF and 2A12, showing that terminal branch identity is not altered by ectopic Grh expression (arrowheads). (D) A stage 16 btl-GAL4/UAS-grh embryo carrying the fusion cell marker esg-lacZ double labelled with antibodies against Grh (red) and ß-gal (green) showing that fusion cell differentiation (yellow; double labelled cells) is not affected. (E,F) Confocal optical section (E,F') and projections (F) of btl-GAL4/UAS-grh;UAS-eGFP embryo labelled for Grh (E; red) and GFP (F,F'; green). The positions of the cell nuclei of the dorsal trunk and dorsal branches in E indicate that they have migrated away from the tracheal sac. The cells extend elaborate basolateral projections (F), but their apical surface does not elongate to form a branch (arrow in F' marks the tip of the short stump that forms instead of the dorsal branch). (G) Confocal projections of stage 16 btl-Gal4/UAS-grh embryo labelled with anti-DE-cad, showing apical cell circumferences and outline of the apical (lumenal) surface in two dorsal trunk metameres. The lumenal cavities fail to elongate and fuse despite the fact that the DT cells migrate to become juxtaposed (see D,E,F). (H,I) Stage 16 wild-type (H) and term-GAL4/UAS-grh (I) embryos carrying the cytoplasmic marker trh-lacZ, labelled with antibodies against ß-gal (red) and 2A12 (green). Ectopic Grh expressed in single terminal cells prevents lumen growth into the cytoplasmic extensions. Terminal branches (TB) are indicated by arrowheads. Scale bars: 10 µm.

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