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doi: 10.1242/10.1242/dev.00218

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Grainy head controls apical membrane growth and tube elongation in response to Branchless/FGF signalling

Johanna Hemphälä1,*, Anne Uv2,*, Rafael Cantera3, Sarah Bray4 and Christos Samakovlis1,{dagger}

1 Department of Developmental Biology, Wenner-Gren Institute, Stockholm University, S-10691 Stockholm, Sweden
2 Department of Medical Biochemistry, University of Gothenburg, S-405 30 Gothenburg, Sweden
3 Department of Zoology, Stockholm University, S-10691 Stockholm, Sweden
4 Department of Anatomy, University of Cambridge, Cambridge, CB2 3DY, UK



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  		Fig. 1. Grh is expressed in the developing trachea and is required to prevent excessive tube extension. (A) Lateral view of the wild-type embryonic tracheal system at stage 16, visualised by antibodies against the 2A12 (lumenal) antigen. The major tracheal branches, dorsal trunk (DT), dorsal branch (DB), lateral trunk (LT), transverse connective (TC) and ganglionic branches (GB) are indicated. The rectangle outlines the part of the trachea that is shown in the subsequent panels, except for in F-I. (B-G) Tracheal lumens of wild-type (B,D,F) and grhs2140/Df(2R)Pcl7B mutant (C,E,G) embryos at stage 16. At the beginning of stage 16, grh mutants (C) have more elongated DTs than wild-type embryos (B). The DT growth continues as stage 16 proceeds, and at the end of this stage (D,E), the excessive tubular extension is evident in additional branches, including the TC (arrowheads in E), LT (not shown) and GB (F,G; ventral lateral view). (H-I) Dorsal view of wild-type (H) and grhs2140/Df(2R)Pcl7B mutant (I) embryos stained for 2A12 and DSRF, showing three pairs of unicellular terminal branches (arrowheads) emanating from the dorsal branches. Terminal branching and DSRF expression is normal in grh mutant embryos, but at the end of embryogenesis, the terminal branches become convoluted and often make loops in grh mutants (I, asterisk). (J,K) Wild type (J) and grh mutants (K) labelled with antisera against the nuclear protein Trh, which is expressed in all tracheal cells. The number of DT and DB cells is not greater in grh mutants than wild type. (L,M) Wild-type and (O,P) grhB37/grhB37 mutant embryos carrying the cytoplasmic trh-lacZ marker were labelled for Grh (red; L,O) and b-Gal (green; M,P). Grh is expressed in all tracheal cells in wild-type embryos (L; stage 14), and is absent in grh mutants (O; stage 14). (N,Q) Wild-type (N) and grhB37 embryos (Q) carrying the Grh activity reporter, GBE-lacZ, were double labelled with mAb2A12 (green) and anti-b-Gal (red). GBE-lacZ is expressed in the wild-type trachea (N; red nuclear staining), but is absent in grh mutants. Scale bar in A: 25 mm; B-K, 10 mm; L-Q, 20 mm. In this and all subsequent figures anterior is left and dorsal up.

 


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  		Fig. 2. Irregular cell shapes, but normal apical-basal epithelial cell organisation in grh mutants. (A,D) Confocal projections of a DT segment of wild-type (A) and grh mutants (D) labelled with antibodies against DE-cad to visualise the apical cell circumference. The tracheal cell shapes in grh mutants are irregular and elongated compared to the wild type. (B,C,E-L) Confocal longitudinal sections of one segment of the DT, to visualise the subcellular localisation of apical, lateral and cytoskeletal markers in wild-type and grh mutant embryos. Embryos carrying the cytoplasmic trh-lacZ marker (B,E) were double labelled with antibodies against ß-gal (green) and Crumbs (red). The levels and subcellular localisation of Crumbs at the apical cell surface is the same in wild-type (B) and grh mutants (E). Double labelling of wild-type (C) and grh (F) embryos with anti-DE-cad (red) and anti-Nrx IV (green) shows that cadherin is localised more apically than Neurexin both in grh mutants and in wild-type embryos. Labelling for ß-heavy spectrin (G,J) shows concentrated localisation at the apical surface of the tracheal cells, both in wild-type (G) and grh mutants (K). Wild-type (H) and grh mutants (J) expressing UAS-Act-GFP in all tracheal cells were labelled with GFP. The apical localisation of actin-GFP is not affected in grh mutants. Wild-type (I) and grh mutants (L) carrying the trh-lacZ reporter were double labelled for ß-gal (green) and Coracle (red). The lateral membrane localisation of Coracle is not affected by grh. Scale bar: 5 µm.

 


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  		Fig. 3. Expanded apical membrane in grh mutants. (A,B) Electron micrographs of the DT (cross section) in wild-type (A) and grh mutants (B) at early stage 17. The lumen is disorganised in grh mutants, with tongues of cell masses extending from the apical surface to occupy part of the lumen. grh mutant embryos can secrete lumenal and cuticular components, but the characteristic taenidial structure is in places disrupted. (C,F) DT cross section at higher magnification showing a single cell of wild-type (C) and a grh mutant (F) with the membranes outlines in black. (D,G) Drawings of single cells based on EM cross sections in C and F. The cell membrane is shown in black, the apical junctions are marked with red, and the apical membrane is blue. In grh mutants, the amount of cell membrane apical to the AJs is significantly greater than in the wild type and excessive membrane folds over neighbouring cells. (E,H) High magnification images focused on a single AJ. In the wild-type cell (E), the AJs are positioned near the apical surface perpendicular to the lumenal surface. In the grh mutant (H), AJs are generally positioned further away from the lumen and are occasionally smaller. Scale bars in A,B, 1 µm; C,F, 0.5 µm.

 


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  		Fig. 4. Tracheal lumen morphology in fasII and ATP{alpha} mutants. (A-F) Lumens (A,B,D,E) and dorsal trunk cell shapes (C,F) visualised with anti-DE-cad of stage 16 fasIIeb112 (A-C) and Atp{alpha} mutants (D-F). (A,C,D,F) Dorsal views; (B,E) ventral views showing the ganglionic branches. (A,D) Embryos mutant for fasII and Atp{alpha} develop long and convoluted DTs reminiscent of those of grh mutants. Tubular dilations in the transverse connectives are indicated by asterisks. (B,E) The lumen of several branches is discontinuous (arrowheads at the GBs). In fasII, (C) and Atp{alpha} (F) mutants the apical cell surface appears elongated and expanded. Scale bar: 20 µm.

 


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  		Fig. 5. FasII and ATP{alpha} are localised to the lateral cell surface and do not affect apical membrane growth. (A,B) Confocal longitudinal sections of the DT of wild-type embryos labelled with antibodies against FasII (A) and ATP{alpha} (B) show that both proteins localise to the lateral cell surface. (C,D) The expression and localization of FasII and ATP{alpha} are not affected in grh mutants. (E,H) Confocal cross sections of the DT of wild-type (E,F) and Atp{alpha} mutant (G,H) embryos labelled with antibodies against DE-cad (red; E,G) and Nrx (green; F,H). The lateral localization of Nrx is diffuse in the Atp{alpha} mutants. (I) Electron micrograph revealing a partial cross section of the DT in an Atp{alpha} mutant embryo at late stage 16. No excessive growth of the apical membrane can be seen in the Atp{alpha} mutants, and the AJs appear correctly localised near the lumenal surface. Scale bar: A-D, 10 µm; E-H, 5 µm; I, 0.5 µm

 


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  		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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  		Fig. 7. Bnl activity modulates GBE-lacZ expression. (A-D) Ectopic Bnl expression in the trachea causes up-regulation of GBE-lacZ expression. Lateral view of a portion of the DT of wild-type (A,B) and btl-GAL4/UAS-bnl (C,D) stage 16 embryos, carrying GBE-lacZ and double labelled for ß-gal (green; A,C) and Grh (red; B,D). GBE-lacZ expression is enhanced in most tracheal cells upon ectopic Bnl signalling (compare A and C), but Grh levels remain the same (compare B and D). (E-H) Ectopic expression of Bnl in single terminal cells results in increased levels of GBE-lacZ expression in this and neighbouring cells. Dorsal lateral views showing part of the DT and two dorsal branches of wild-type (E,F) and Term-GAL4/UAS-bnl (G,H) embryos carrying GBE-lacZ, and labelled for ß-gal (green, E,G), and lumenal antigen 2A12 and DSRF (both in red; overlaid with green in F and H). Ectopic Bnl signalling in terminal cells results in the expression of the terminal marker DSRF in additional cells (H) and in the same cells the level of GBE-lacZ expression is increased (G,H). (I-L) Bnl is required for GBE-lacZ expression. Lateral view of two tracheal segments in embryos mutant for pnt (I,J) and bnl (K,L) that carry GBE-lacZ. Double labelling for 2A12 (red) and ß-gal (green) shows that tracheal GBE-lacZ expression is reduced in bnl mutant embryos (K,L) as compared to the epidermal expression, or to the tracheal expression in pnt mutants (I,J), which represents wild-type levels. Scale bars: 10 µm.

 

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