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Fig. 7. ash2 regulates kni expression. kni in wild-type (A), ash2^I1/ash2^I1 (B) and ash2¹¹²⁴¹¹/ash2¹¹²⁴¹¹ (C) wing discs. (D) Minute⁺ clones homozygous for ash2^I1 induced at 110±4 hours AEL are marked by the absence of ß-galactosidase. (E) Expression of kni is cell-autonomously upregulated by the loss of ash2 function (arrow). There is residual kni expression in heterozygous tissue. (F) Merged images of C and D. (G) Clones homozygous for ash2^I1 induced at 52±4 hours AEL are marked as black spots by the absence of GFP and twin clones of wild-type cells appear as intense green spots. (H) kni expression is upregulated in homozygous ash2^I1 mutant cells and residual kni disappears in cells homozygous for GFP. (I) Merged green and red channels of F and G. (J) Detail of a clone. Homozygous GFP cells are outlined in white (HMZ) and homozygous ash2^I1 cells are outlined in yellow. HTZ. heterozygous tissue. (K) Anti-Kni staining of the same disc. One mutant copy of ash2 is enough to de-repress kni expression. (L) Merged images of I and J, and an additional channel with nuclear labelling (propidium iodide, shown in blue). (M) Detail of endogenous Kni (red) showing nuclear localisation (blue) in the L2 domain (pink in lower panel corresponds to nuclear labelling). (N) Detail of an ash2^I1 clone; Kni is mainly cytoplasmic, although nuclei also contain some protein. (O) Adult kni^ri–1/kni^ri–1 wing. (P) kni^ri–1/kni^ri–1 ash2¹¹²⁴¹¹/ash2¹¹²⁴¹¹ wing showing rescue of L2 vein. (Q) rho expression in pupal kni^ri–1/kni^ri–1 wing. (R) rho expression in pupal kni^ri–1/kni^ri–1 ash2¹¹²⁴¹¹/ash2¹¹²⁴¹¹ wing. There is distal expression in L2. (S) RT-PCR showing increased kni mRNA in ash2^I1 homozygous larvae compared with wild type. Rp49 is shown as a control. Scale bars: 20 µm.

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