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doi: 10.1242/10.1242/dev.00535

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Novel level of signalling control in the JAK/STAT pathway revealed by in situ visualisation of protein-protein interaction during Drosophila development

Stephen Brown ^?,*, Nan Hu ^?,* and James Castelli-Gair Hombría^?,

^? Department of Zoology, University of Cambridge, Downing Street, Cambridge CB2 3EJ, UK
^* These authors contributed equally to this work

View larger version (108K): [in a new window]   Fig. 1. Homo-dimerisation of DOME as detected by the ßlue-ßlau technique. (A) Wild-type posterior spiracles with an elongated filzkörper (arrow). (B) Zygotic phenotype of dome217, the filzkörper is not formed. (C) Rescue of dome217 with UAS-dome. Expression of the hybrid receptor in the spiracles using Klu-Gal4 rescues the filzkörper, showing that the hybrid protein is functional. Optical sections through the salivary gland (D,F,H) and the hindgut (E,G,I). White arrowheads point to the apical membrane, black arrowheads to the basal membrane on one side of the tube. Note that the salivary gland is closed at one end whereas the gut tube is open at both ends. (D-E) Anti-ß-gal staining of UAS-dome, UAS-dome expressed using h-Gal4. The hybrid receptors localise mainly to the apical membrane, although the proteins are also detected in the cytoplasm. (F-G) X-gal staining of the same genotype as in D-E. The blue precipitate is formed at the apical side of the cell in both the salivary glands and the hindgut showing that the product of the reaction has a limited diffusion through the cell. (H-I) X-gal staining of UAS-dome expressed using h-Gal4. No coloured precipitate is seen when only one of the two fusion proteins is expressed, proving that the reaction requires complementation of the hybrid receptors.

View larger version (90K): [in a new window]   Fig. 2. Developmental regulation of receptor dimerisation. Dimerisation in embryos simultaneously expressing UAS-dome and UAS-dome, driven by either da-Gal4 (A-F), 69B-Gal4 (G-J), h-Gal4 (K-L) or 24B-Gal4 (M-N). Left panels show expression of the hybrid protein detected using an anti-ß-gal antibody, right panels show the X-gal patterns induced in those embryos by hybrid protein dimerisation. da-Gal4 and 69B-Gal4 embryos express ß-gal proteins in all ectodermal derivatives but they show X-gal staining in a subset of those cells (Right panels). h-Gal4 drives expression in stripes (K) but only the tracheal pits in each stripe show X-gal staining (L). Arrows in K-L point to tracheal pits where the hybrid proteins are expressed, and white arrowheads to a tracheal pits without expression. The 24B-Gal4 line drives high levels of expression in the mesoderm (M) but no X-gal expression is observed in this tissue (N). (A-B,K-N) Stage 11 embryos, (C-J) stage 13-14 embryos. When comparing embryos at the same developmental stage, all ectoderm Gal4 lines induce comparable X-gal staining (compare B with L, D with H and F with J). as, amnioserosa membrane; hg, hindgut; ms, mesoderm; ps, posterior spiracle; sg, salivary glands; tp, tracheal pits.

View larger version (103K): [in a new window]   Fig. 3. Effects of altered ligand expression on receptor dimerisation. Expression of UAS-dome and UAS-dome (abbreviated as UAS-ßß in the Fig.) was induced by da-Gal4. X-gal stainings show patterns of receptor dimerisation. (A-B) Embryos in which ectopic ligand expression has been induced using UAS-upd do not show ectopic X-gal staining (compare with Fig. 2B,D). (C-D) Patterns of X-gal staining are not affected in embryos deficient for upd. (E-F) Expression of a dominant negative receptor does not affect patterns of dimerisation.

View larger version (69K): [in a new window]   Fig. 4. Effects of altered ligand expression on embryo development. (A) Cuticle of a late embryo showing the result of ectopic UPD expression. There is abnormal retraction of the germ band and a dorsal hole (arrowhead) indicating abnormal development of the amnioserosa, whereas there is little effect on the denticle belts. (B-C) Expression of kni in the wild type (B) and in embryos with ectopic UPD expression (C). (D-F) Expression of trh in the wild-type (D), upd– (E) and ectopic UPD embryos (F). Although the JAK/STAT pathway is required for trh expression (E), ectopic upd cannot activate trh expression outside the tracheal pits (F). (G-I) Expression of vvl in the wild-type (G), upd– (H) and ectopic UPD embryos (I). In the wild type, expression of vvl in the hindgut is restricted to the small intestine (arrowhead in G). vvl is not expressed in the hindgut (arrowhead) of ligand-deficient mutants (H). Ectopic upd results in ectopic vvl activation in the hindgut (arrowhead in I). Df(1)os 1A was used as a deficiency for upd and related genes. (A-F) Lateral views, (G-I) dorsal views. Anterior is left in all panels. (A) Dark field image of a late embryo cuticle. (B-F) Stage 11 embryos. (G-I) Stage 13 embryos.

View larger version (26K): [in a new window]   Fig. 5. Alternative models for JAK/STAT receptor complex-ligand interaction. In A the monomeric receptors exist in the inactive state. Ligand binding induces receptor dimerisation and activation. If this model was correct, ectopic ligand expression should result in ectopic blue staining because of ectopic dimerisation, whereas deletion of the ligand should abolish the blue staining. In the alternative model (B), the inactive receptors are dimerised. Ligand induction activates the receptors through conformational modification. If this model is correct, blue staining would be independent of the expression patterns of the ligand. Experiments in Fig. 3 support the second model. Green ovals, cytokine binding module; red ovals fibronectin type III repeats of DOME. The intracellular receptor domain (orange) binds to HOP (JAK). Blue boxes represent and ß-gal mutants. Complementation of the mutant proteins occurs when receptor dimerisation brings them into close proximity.