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First published online 26 February 2008
doi: 10.1242/jcs.019422

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Grainy head promotes expression of septate junction proteins and influences epithelial morphogenesis

¹ Wellcome Trust/Cancer Research UK Gurdon Institute of Developmental Biology and Cancer, University of Cambridge, Downing Street, Cambridge, CB2 3DY, UK
² Department of Physiology Development and Neuroscience, University of Cambridge, Downing Street, Cambridge, CB2 3DY, UK
³ Department of Biological Sciences, Tata Institute for Fundamental Research, Colaba, Mumbai 400 005, India
⁴ Institute för Medicinsk och Fysiologisk Kemi, Medicinaregatan 9A, Göteborgs Universitet, Göteborg, Sweden

View larger version (39K): [in this window] [in a new window]   Fig. 1. Expression of Grh in the amnioserosa perturbs the progression and outcome of dorsal closure. (A,B) Dorsal closure: a membrane-associated Myc-tag labels amnioserosa at the stages indicated (lateral view; c381::Gal4). Arrows mark the edge of the epidermal sheets. (C,F) Grh expression in wild-type (C) and ASG332>grh (F) embryos (arrows indicate amnioserosa; stage 14, lateral view). (D,G) Phalloidin staining marks the cell outlines in dorsal view of wild-type (D) and ASc381>grh (G) stage-15 embryos; zippering has commenced at the poles in control but not in ASc381>grh embryos. Arrows mark the edge of the epidermal sheets. (E,H) Cuticles from hatching stage. ASc381>grh embryos (H) have a dorsal hole (arrows) and internal organs are extruded (asterisk).

View larger version (70K): [in this window] [in a new window]   Fig. 2. Grh promotes expression of SJ components in the amnioserosa. Stage-14 control (A,C,E-I,M,N) and ASc381>grh (B,D,J-L,O,P) embryos stained as indicated. Dorsal views of whole embryos (A-D) or of amnioserosa (E-L, single confocal sections; J-J'' are individual channels from one embryo). (M-O) Transverse sections stained as indicated to reveal the apical/basal distributions. (M,O) Upregulated Fas3 (FasIII) in ASc381>grh embryos accumulates apically (green in O, white in O'; e.g. orange arrowheads). (N,P) Dlg is also upregulated in ASc381>grh embryos (green, P; white P') and spreads along the apical surface (e.g. orange arrowheads). (M-P) White arrows mark lateral junction sites.

View larger version (89K): [in this window] [in a new window]   Fig. 3. fas3 and coracle are potential targets of Grh. (A,B) Levels of Fas3 (FasIII; red, A,B; white, A',B') and Coracle (blue, A,B; white, A'', B''') are subtly reduced in grh-mutant clones (identified by absence of GFP, green, in A,B). Clone boundaries are indicated by arrows. (C) Grh binds to sites from fas3 and coracle genes. Amount of Grh complexed with labelled Gbe2-binding sites in the absence (O) or presence of the indicated competitors (5x molar excess and 50x molar excess). N-box is an unrelated E(spl) target site. Lane 1 has no added protein; all other lanes contain GST-Grh bacterial extract. (D) Fold stimulation by Grh of luciferase reporter genes containing fragments spanning the indicated sites. 4xGbe, positive control with oligomerised Gbe2 sites; hid1, negative control. (E) Rhodamine-conjugated dextran is excluded from the tracheal lumen (arrows) of wild-type (wt) and grh-mutant (grhB32/grhB32) embryos but not from SJ mutants (Atp; SJ–).

View larger version (51K): [in this window] [in a new window]   Fig. 4. Grh expression results in altered cellular morphology and disrupts the distribution of integrins and cadherin. Dorsal views of control (A,B) and ASc381>grh (C-F) embryos stained with antibodies against βPS integrin (green, A-D; white, A'-D') and E-cadherin (magenta, A-E; white, A''-D'',E',F). (A-D) ASc381>grh cells have poor localisation and patchy accumulation of βPS integrin (e.g. arrows, C,C'), reduced βPS integrin levels at epidermal interface (arrowheads, C,C',D,D'), reduced co-localisation between βPS integrin and E-cadherin (compare B with D), and some mislocalisation of E-cadherin (e.g. arrows C''). (E,F) Defects in the epidermis include bunching (orange arrows) and splaying (green arrows) of epidermal cells, and a detachment between the epidermis and the amnioserosa. (E) Nuclei are visualised with DAPI.