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First published online March 4, 2005
doi: 10.1242/10.1242/dev.01689

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Differential regulation of Hedgehog target gene transcription by Costal2 and Suppressor of Fused

Karen S. Ho^*, Kaye Suyama, Matthew Fish and Matthew P. Scott

Departments of Developmental Biology and Genetics, Howard Hughes Medical Institute, Clark Center W252, Stanford University School of Medicine, 318 Campus Drive, Stanford, CA 94305-5439, USA

View larger version (82K): [in a new window]   Fig. 1. Effect of different mutants of Cos2 on dpp-lacZ expression. (A,B) dpp-lacZ reporter expression at the AP boundary in wild-type third instar larval discs. Nuclear ß-galactosidase is immunofluorescently labeled (red). Area in A (white box) is shown at higher magnification in B. (C,D) Overexpression of cos2GFP using the 71B Gal4 driver (green) represses dpp-lacZ expression (red) at the AP boundary. (E,F) A small FLP-out clone expressing Motor-GFP (green) in the anterior compartment ectopically expresses dpp-lacZ (red) in a cell-autonomous manner. A region of normal dpp-lacZ expression at the AP boundary is shown (arrowhead). (G,H) An anterior FLP-out clone of cells expressing Neck-GFP (green) ectopically expresses dpp-lacZ (red) in a cell-autonomous manner. (I,J) Cos2C-GFP expression (green) driven by 71B Gal4 represses dpp-lacZ expression (red) at the AP boundary. (K,L) A large FLP-out clone expressing S182N-GFP derepresses dpp-lacZ expression within the clone (red) in a cell-autonomous manner. Box in K indicates the area that is shown at higher magnification in L. The normal expression of dpp-lacZ at the AP boundary is shown in K (yellow arrowhead). Overgrowth of anterior tissue caused by the clone is indicated (white arrow). A posterior clone, which does not ectopically express dpp-lacZ or produce overgrowth of tissue, is also indicated (blue arrowhead). Because not all nuclei in the disc lie in the same optical plane, there appear to be variations in dpp-lacZ staining in different parts of the disc. Correcting this by focusing on small local areas of the disc confirms that dpp-lacZ is expressed at uniform, high levels throughout S182N-expressing clones (data not shown). (M,N) A FLP-out clone expressing S182T-GFP at the AP boundary (green) that interrupts the normal region of dpp-lacZ expression represses dpp-lacZ expression (red) in a cell-autonomous manner. Box in M indicates the area that is shown at higher magnification in N. (O) A chart summarizing the effects each mutation has on dpp-lacZ expression in either the anterior compartment of the disc or at the AP boundary where dpp-lacZ is normally expressed. +++ indicates high uniform levels of derepression or repression. Also shown are schematic drawings of the putative Cos2 homodimer with appropriate alterations to reflect each mutation or deletion. Results were the same for each construct and its C-terminally fused GFP counterpart except for Motor, for which Motor-GFP expressing flip-out clones could not be generated. For this and all other figures, wing discs are oriented such that anterior is leftwards and dorsal is downwards.

View larger version (72K): [in a new window]   Fig. 2. Wing duplications resulting from S182N expression can be suppressed by co-expression of cos2. (A) Wild-type wing (normal costa indicated by arrow). (B,C) Examples of the two extremes of expressivity seen in wing blades in which S182NGFP is expressed using MS1096 Gal4. The same results are seen in wings expressing S182N. The outgrowth of the costa is shown in B by an arrow. B represents the very mild phenotypes seen, while C represents the extreme phenotypes seen. (D) Overexpression phenotype of MS1096 Gal4; UAS S182T/+ flies. Wing blades are reduced in size and third and fourth wing veins are missing. (E) Phenotype of MS1096 Gal4; UAS cos2GFP/+ flies. There are no signs of anterior wing outgrowth or duplications, but there is the similarity between E and D. (F) Phenotype of MS1096 Gal4; UAS S182N/+; UAS cos2/+ flies. Note there are no signs of anterior wing outgrowth or duplications, and the phenotype is similar to E and D. All flies were grown at 29°C to maximize levels of expression using the MS1906 Gal4 driver, which strongly drives expression throughout the wing pouch. All wings are photographed at the same magnification to show the differences in wing size between each genotype.

View larger version (31K): [in a new window]   Fig. 3. The difference in S182N and Cos2 activities is not due to differences in expressed protein levels. Western blot showing overexpression of mutant and wild-type cos2 results in production of comparable levels of each protein in imaginal wing disc lysates (30 µg protein loaded per lane). Arrows indicate migration differences between GFP-tagged proteins and their untagged counterparts. The overexpressed protein is indicated above each lane on the blot. WT, wild-type lysates; Cos2, Hh indicates overexpression of both Cos2 and Hh; S182N-GFP, Hh indicates over-expression of both Hh and S182N-GFP. All experiments were performed using independently isolated fly lines of tagged and untagged Cos2 proteins with the same results in all experiments. 71B Gal4 was the driver for all constructs. BAP111 is a ubiquitous nuclear protein used as a loading control.

View larger version (38K): [in a new window]   Fig. 4. Expression of S182N stabilizes Ci in its unprocessed, 175 kDa form in wing imaginal discs. (A-D) Stabilization of Ci shown by immunostaining of discs using a monoclonal antibody recognizing the unprocessed form of Ci only (CiFL). (A) A wild-type disc showing normal levels of stabilization of Ci at the anteroposterior boundary. CiFL signal is strongest along the AP boundary (bracket). (B) Over-expression of Cos2 using the 71B Gal4 driver results in reduced levels of CiFL at the AP boundary when compared with wild type (bracket). (C) Expression of S182N using the 71B Gal4 driver results in a wider stripe of cells at the AP boundary positive for CiFL expression (bracket). Cos2-GFP expression driven by 71B Gal4 is shown in D. The same expression pattern is achieved for the expression of S182N driven by 71B Gal4 (not shown). (E) A protein blot of wing imaginal disc extracts probed with anti-Ci antibody 1C2 (a gift from Robert Holmgren), which recognizes both processed (75 kDa) and unprocessed (175 kDa) forms of Ci. Lane 1, wild-type imaginal disc extract; lane 2, extract from discs overexpressing Hh; lane 3, extract from discs overexpressing Cos2; lane 4, extract from discs simultaneously overexpressing Cos2 and Hh; lane 5, extract from discs overexpressing S182N; lane 6, extract from discs overexpressing S182T Cos2. 71B Gal4 was used to drive expression of all transgenes indicated. (F) Quantitation of three independent western blots is plotted as relative amounts of CiR to CiTOTAL (y-axis) in wing disc lysates corresponding to genotypes expressing the UAS transgenes indicated driven by 71B Gal4 (x-axis).

View larger version (42K): [in a new window]   Fig. 5. Genetic removal of Su(fu) activity changes S182N Cos2 from a repressor of ptc transcription to an activator. (A,F) Expression of ptc-lacZ (red) in a wild-type (A), and Su(fu) homozygous mutant background. (B,C) MS1096 Gal4-driven S182N-GFP (green) represses normal ptc-lacZ expression at the AP border (red) in a wild-type background. (G,H) By contrast, expression of S182N-GFP (green) causes the derepression of ptc-lacZ (red) in anterior cells of a Su(fu)/+ mutant wing disc. (D,E) Expression of S182T Cos2-GFP (green) by 71B Gal4 represses normal ptc-lacZ (red) expression at the AP boundary in a wild-type background. (I,J) Expression of S182T Cos2-GFP (green) by MS1096 Gal4 represses ptc-lacZ (red) expression at the AP boundary and no ectopic ptc-lacZ expression is observed in the anterior cells of a Su(fu)/+ mutant wing disc.

View larger version (22K): [in a new window]   Fig. 6. Su(fu) protein is phosphorylated in response to Hh overexpression. (A) Western blot of extracts from imaginal wing discs probed with anti-Su(fu) antibody. BAP 111 was used as a loading control. Lane 1, extract from wild-type discs; lane 2, extract from Hh overexpressing discs; lane 3, extract from Cos2-overexpressing discs; lane 4, extract from discs expressing S182N; lane 5, extract from discs expressing S182T; lane 6, extract from discs overexpressing both Hh and Cos2 simultaneously; lane 7, extract from discs overexpressing both Hh and S182N simultaneously. Overexpression of proteins was achieved in each case by using the 71B Gal4 driver. Arrowheads indicate the migration of singlet and doublet bands recognized by the Su(fu) antibody. The Su(fu) doublet appears only in those lanes in which Hh has been overexpressed. (B) Treatment of wing disc lysates with the lambda phosphatase enzyme, which removes phosphates from both Ser/Thr and Tyr, shifts the doublet Su(fu) band to a single band that co-migrates with Su(fu) from wild-type lysates. Lane 1, extract from 71B Gal4 wild-type discs, showing a single band for Su(fu); lane 2, the Su(fu) doublet is seen in lysates from Hh-overexpressing discs; lane 3, treatment of 71B Gal4 wild-type lysates with lambda phosphatase does not change the migration properties of Su(fu), compare lane 3 with lane 1, suggesting that the majority of Su(fu) does not exist in a phosphorylated state in wing imaginal discs. Lane 4, treatment of Hh-over-expressing lysates with lambda phosphatase shifts the Su(fu) doublet (Lane 2) to a singlet (arrowheads). Dsh, Dishevelled protein, recognized by Rabbit anti-Dsh (gift from Karl Willert and Roel Nusse), was used as a control as it normally exists as a hyperphosphorylated protein (lane 1). The successful dephosphorylation of Dsh can be seen by a change in its migration as multiple bands (lanes 1 and 2) to a single major band (lanes 3 and 4).

View larger version (90K): [in a new window]   Fig. 7. cos2 mutant clones and S182N-expressing clones at the AP boundary do not express anterior en. (A-D) cos2- cells in a clone at the AP boundary lacks anterior en expression. A disc stained with anti-Myc (red) to reveal cos2- clones, which are not Myc-positive and are therefore lack red signal (arrow), is co-stained with anti-En 4D9 (green). The area marked in A (white box) is shown at higher magnification in B-D. No En is detected within the clone (arrow). (E-G) Cells at the AP boundary expressing S182N-GFP (green) fail to express anterior En (blue, arrow). The area shown in E (white box) is shown at higher magnification in E and F.

View larger version (34K): [in a new window]   Fig. 8. Differential gene regulation by Hedgehog (Hh). Hh protein, secreted from posterior compartment cells (green), is distributed as a protein gradient in anterior compartment cells, with the highest concentration of Hh occurring at the AP boundary (black line). Cells receiving minimal amounts of Hh respond by inactivating the ATPase activity of Cos2 (red domain), perhaps by Fu-dependent phosphorylation (Nybakken et al., 2002) or other event. This results in the activation of dpp transcription in cells as far as 15 cell diameters away from the Hh source (red nuclei - only eight cells shown for sake of brevity). Cells receiving an intermediate amount of Hh inactivate Su(fu) (purple domain), perhaps by phosphorylation, which results in the activation of ptc (purple nuclei) in addition to the activation of dpp. ptc is expressed in a swath of cells 8-10 cell diameters in width (only five cells shown). Anterior en transcription in a 5- to 7-cell-wide stripe is Hh dependent and is located closest to the AP boundary (only one cell shown, labeled En). Posterior En protein (En) is also present but its expression is not Hh dependent. In the anterior, both dpp and ptc transcription are repressed by en (Sanicola et al., 1995), so lower amount of dpp transcription is seen in a 1- to 5-cell-wide stripe at the AP border in en-expressing cells (white nucleus of anterior cell labeled En).