Figure 3. tkv heterozygous mosaicism disrupts wing pattern formation.

(A–D) Germline inheritance of the recessive tkv^C97R-mCherry mutation (A). Tkv^C97R-mCherry (red) and p-Mad (grey) expression in a heterozygous wing disc (B, C). tkv^C97R heterozygotes developed normal wings (D). Positions of longitudinal vein L2-L5 are shown. (E–J) Generating somatic clones of tkv^C97R heterozygous cells by heat shock (E). wild-type and tkv^C97 heterozygous cells are shown by green and red, respectively (F, G, I, J). tkv^C97R-mCherry heteromosaicism disrupted the p-Mad activity gradient (grey) and caused wing vein patterning defects (H). (I, J) Magnified images of the boxed area in (F). A similar effect on p-Mad activity was observed in male wing discs, although adult males exhibited milder wing phenotypes. Scale bars: 50 μm for (B), 0.5 mm for (D), and 10 μm for (I). Anterior is oriented to the left side of wing disc images and to the top side of adult wing pictures. (K) Quantification of adult wing phenotypes in tkv^C97R-mCherry heterozygous and heteromosaic animals. Longer heat shock generated more uniformly heterozygous cell populations and reduced mosaicism, rescuing wing vein phenotypes.

Figure 3—figure supplement 1. Basal extrusion of tkv^C97R homozygous mutant clones from the wing epithelia.

(A–D) tkv^C97R homozygous mutant clones were basally eliminated from wing epithelia. (A, B) To generate mCherry-labelled wild-type control clones, hs-flp; tkv^GFP>>mCherry/tkv^mCherry animals were heat shocked for 10 min at 72 hr AEL. (C, D) Homozygous mCherry-labeled tkv^C97R clones were generated with an identical heat shock regimen in larvae of the genotype: hs-flp; tkv^{GFP>>C97R-mCherry}/tkvC^C97R-mCherry. The upper and lower panels show standard XY and optical cross section XZ images of each wing disc, respectively. Arrowhead (red) indicates eliminated tkv^C97R homozygous mutant cells. Scale bars: 50 μm. Anterior is left.

Figure 3—figure supplement 2. tkv heteromosaicism does not induce cell death.

(A–D) Cell death was not observed in wing discs possessing tkv^C97R heteromosaic clones. Wing discs from hs-flp; tkv^GFP>>mCherry/+ (control, A, B), and hs-flp; tkv^{GFP>>C97R-mCherry}/+ (C, D) animals heat shocked at 72 hr AEL for 10 min were stained with anti-cleaved Drosophila Dcp-1. Scale bars: 100 μm. Anterior is left.

Figure 3—figure supplement 3. Additional JPS-associated tkv mutations exhibit deleterious heteromosaicism.

(A–F) Deleterious mosaic effects of tkv^C40Y and tkv^M442T. Wing discs from wild-type control (A, B), tkv^{GFP>>C40Y-mCherry} (C, D), and tkv ^{GFP>>M442T-mCherry} (E, F) animals. Tkv-GFP and Tkv*-mCherry are depicted by green and red, respectively. tkv^C40Y and tkv^M442T heteromosaic resulted in aberrant p-Mad expression (grey, C–F). (G–L) Context dependent Tkv^C90R activity. The hinge cells expressing Tkv^C90R-mCherry induced ectopic p-Mad expression (grey, orange box in G), (I, J), while the same mutation showed a weaker signaling capability in the wing pouch (green box in G), (K, L). Red and green arrowheads indicate locations of tkv^C90R heterozygous and wild-type cells, respectively. Scale bars: 50 μm in (A) and (G), 10 μm in (I) and (K). Anterior is left.

Figure 3—figure supplement 4. Effects of tkv^C97R heterozygous mutant clones in the developing eye and haltere discs.

(A–Q) tkv^C97R heterozygous clones in the developing eye (A–I), and haltere discs (J–Q). For these experiments, hs-flp; tkv^GFP>>mCherry/+ (control, (A, B, J, K), and hs-flp; tkv^{GFP>>C97R-mCherry}/ + (experimental, (D, E, M, N) animals were heat shocked at 72 hr AEL for 10 min and discs were stained with anti-p-Mad antibody. The boxed areas in (D and M) are shown at higher magnification in (G) and (H), and (P) and (Q), respectively. While p-Mad levels in both discs were mildly disrupted, animals carrying tkv^C97R heterozygous clones developed normal eyes and halteres. Scale bars: 50 μm (A, J), 0.2 mm (C), 10 μm (G, P), 100 μm (I, L).