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Fig. 8. Phosphorylation of Cos2 on Ser572 is necessary for anterior Engrailed expression. (A) 71BGAL4/UAS cos2-WT-myc, (B) 71BGAL4/UAS cos2-572A-myc, (C) 71BGAL4/UAS cos2-572D-myc and (D) yw,hs-flp; FRT42D cos2^W1/FRT42D armlacZ imaginal discs are stained for En (4D9) in blue and Myc in green, except in D,D' in which ß-gal is in green. (A'-D') En is in white and an enlargement of the squared region is shown; the bracket indicates the anterior region at the anteroposterior (AP) boundary. (E,E') yw,hs-flp; FRT42D cos2^W1/FRT42D armlacZ;ac71BGAL4/UAS cos2-WT-myc, (F-G') yw,hs-flp; FRT42D cos2^W1/FRT42D armlacZ; 71BGAL4/UAS cos2-572A-myc and (H,H') yw,hs-flp; FRT42D cos2^W1/FRT42D armlacZ; 71BGAL4/UAS cos2-572D-myc imaginal discs are stained for Myc (red), ß-gal (green) and En (blue). (E''-H'') En is in white and enlargements of the cos2^W1 clones pointed to by an arrow are shown. (A) In the anterior region, Cos2-WT transgene expression (18°C) is sufficient to strongly inhibit the endogenous expression of En. (B) The two mutant Cos2 variants are less efficient, because en expression is only slightly reduced. (D) Cos2 is necessary for the high level of Hh signalling, because En expression is lost (arrow in D') in anterior cos2^W1 loss-of-function clones situated at the AP boundary. (E-H) En expression in anterior clones situated inside the 71B domain at the AP boundary was observed in different discs from distinct experiments (n clones=11 to 15). The rescue domain is visualised by Myc staining and encircled in E-H. cos2^WI loss-of-function clones are identified by loss of ß-gal staining, and anterior clones near the AP boundary are encircled by an unbroken line (E'-H'), or arrows (E''-H''). En expression is absent in cos2^W1 loss-of-function clones expressing the cos2-WT and cos2-572D transgenes. Expression of the cos2-572A transgene gave similar results in 60% of the discs (F-F''), but in the other 40%, a low level of En was detected in cos2^W1 loss-of-function clones in which Cos2-572A was expressed at a low level (G-G''). Notice that the level of En is much higher in the few anterior cells outside of the cos2 mutant clone (arrowhead in G''). Notice also that some cells in the cos2^W1 clones (G'') showed no En expression due to the higher expression of the transgene in these cells.

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