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Fig. S1. Discs lacking PE were able to elongate at the onset of metamorphosis. Surface view and a z-section (inset) of a Ubx>Lines wing imaginal disc at 6 hours APF. Wings lacking PE failed to contribute to adult structures. However, the epithelium of these discs elongated at the onset of metamorphosis from a pseudo-stratified epithelium, in which nuclei were detected at different planes and formed a thickened epithelial layer, to a simpler epithelium in which nuclei were arranged in one to two cell layers.
Fig. S2. Broad expression of lines with several peripodial drivers replaced the PE with DP. (A,C,E) Discs expressing UAS-GFP using Ubx-GAL4, C311-GAL4 and Tsh-GAL4, respectively. Optical sections were taken at the level of the PE but GFP expression is also detected in the superficial layer of the DP in C and E. Ubx>GFP expression is restricted to the central regions of the PE. C311>GFP is expressed at high levels in a broader domain in the PE, and at lower levels in the DP. Tsh-GAL4 is expressed broadly in the PE, the notum and the proximal hinge. (B,D,F) Discs expressing UAS-Lines with Ubx-GAL4, C311-GAL4 and Tsh-GAL4, respectively, and stained for Nub (red). Arrowheads in B,D,E indicate sectioned plane shown in insets. Embryos were raised at 18°C for 36 hours After egg laying (AEL) to minimize embryonic lethality associated with ectopic gene expression during embryogenesis, and shifted to 25°C for the remainder of larval development. Under these conditions, the flattened morphology of wild-type wing discs (A,C,E) was replaced with a spherical morphology (B,D,F). The replacement of the thin PE with a mirror image duplication of the thickened pseudostratified CE that forms the DP. Also note the mirror image duplication of Nub expression in these discs (insets in B,D,F). Experimental discs that were raised at a constant temperature of 25°C were further reduced in size and often totally lost. Expression of UAS-Lines with Coro-GAL4 had no effect on disc development. Expression of UAS-Lines with Odd-GAL4 was embryonic lethal. Expression of UAS-Lines with the DP-specific drivers MS1096-GAL4, Omb-GAL4 or Sd-GAL4 caused no phenotypes (not shown).
Fig. S3. dpp expression is maintained in wing discs lacking PE. dpp is expressed in a central stripe along the AP compartment boundary in both (A) wild-type and (B) Ubx-Lines discs that lack PE. Opposing arrows in A and B point to the stripe of dpp expression along the AP compartment boundary. Asterisk in B indicates a non-specific background stain.
Fig. S4. Bowl activity in linesRNAi clones reprograms pouch clones to adopt proximal hinge identity. To determine whether the lines clonal phenotype was due to the stabilization of Bowl, we have generated linesRNAi-expressing clones lacking bowl function using the MARCM technique and examined the morphology and the expression of Wg in the clones. (A) lines RNAi MARCM clones; the Wg protein accumulated ectopically in the clones near the wing margin and at the periphery of the wing pouch. We note, however, that Wg expression levels were more variable than these found in lines mutant clones. In some cases, the clones interrupted Wg expression along the wing margin. (B,C) bowl lines RNAi MARCM clones; Wg accumulation was either reduced (B) or missing (C). The lines RNAi clones were qualitatively more circular and tended to protrude basally. The circularity and the protrusive behavior of the clones were qualitatively suppressed in bowl lines RNAi clones. This epistasis experiment further supports the idea that the stabilization of Bowl in lines clones account for their clonal phenotype.
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