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First published online August 2, 2005
doi: 10.1242/10.1242/dev.01927

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Regulation of cellular plasticity in Drosophila imaginal disc cells by the Polycomb group, trithorax group and lama genes

Ansgar Klebes^1,*, Anne Sustar², Katherina Kechris¹, Hao Li¹, Gerold Schubiger² and Thomas B. Kornberg^1,

¹ Department of Biochemistry and Biophysics, University of California, San Francisco, CA 94143, USA
² Department of Biology, University of Washington, Seattle, WA 98195, USA

View larger version (133K): [in a new window]   Fig. 1. Whole-mount in situ hybridization to imaginal discs. Wing and leg discs were hybridized with probes derived from genes predicted to have elevated expression in wing discs (A-J) or leg discs (A'-J'). (A) Ect3 probe stained distinct cell clusters in the presumptive dorsal hinge region of the wing disc; (B) pdm2 probe labeled cells surrounding the pouch with preferential expression in anterior cells; (C) Phk-3 probe labeled several domains in the hinge and notum region; (D) CG4914 expression is enriched throughout the wing disc with highest levels in the dorsal-most region. (E) regucalcin probe produced signal in a population of hemocytes that are associated with the wing imaginal disc, but not the leg imaginal discs (E'). Except for Phk-3, which is expressed in a distinct cell cluster in a ventral position of leg discs, these `wing-specific' genes did not hybridize at detectable levels to leg discs (A',B',D',E'). rho, fd96CB, unc-4 and CG7447 generated more intense staining in leg discs (F',G',H',J'). rho and unc-4 are also detected in wing discs (F,H), whereas no signal above background could be detected for fd96Cb and CG7447 in wing discs (G,J). Dorsal is upwards for all discs; anterior is leftwards.

View larger version (59K): [in a new window]   Fig. 2. Novel markers of transdetermining cells in Wg-expressing leg discs. (A) Schematic representation of a leg disc and the cuts performed to isolate transdetermining (TD, green), dorsal (D, DWT, DWg, orange) and ventral cells (V, VWT, VWg, blue). The area of the weak point is indicated by the dark-green circle. (B,C,D,F,G) In situ hybridization in wg-overexpressing leg discs. CG14059 (B), CG4914 (C) and CG12534 (D) are expressed in the area of the weak point. (E) A cultivated 3/4 fragment of a wild-type 1st leg disc. In the area of the weak point, Vg (E) and Ap protein (E') are both expressed in the dorsal region (E*, merged image: Vg, green; Ap, red) that is similar to the expression domain of CG14059; the expression domain of CG12534 is broader. Expression of lim1 RNA in normal (F) and a Wg-expressing leg disc (G). The distal expression domain of lim1 expands in the Wg-expressing disc (arrowheads) but is absent from an adjacent proximodistal region (outlined). Wg expression causes overgrowth of the dorsal region.

View larger version (70K): [in a new window]   Fig. 3. Expression profiling analysis of transdetermining cells. Cluster analysis of eight different comparisons. Expression ratios are color coded as indicated. Each of 35 columns represents an array experiment grouped into categories as described in Table S1 (see supplementary material). Categories 4, 5 and 5 compare TD cells with DWg, VWg and DWT, respectively. Prior to cluster analysis, the expression ratios were filtered at two levels: (1) spots were required to have intensities with a sum of medians greater than 350; and (2) ratios greater than 2 (1 log2) in at least five out of the 35 experiments. The calculation of self-organizing maps and hierarchical clustering produced a cluster with several sub-clusters, of which six are represented; labeled I-VI with I-IV including genes enriched in TD cells and V and VI containing genes depleted in TD cells. Genes that segregate to sub-cluster I had high levels of expression in TD cells (yellow) and include lama and CG14059. The many absent calls (grey) in the wing-to-leg comparison for genes of this sub-cluster suggest that these genes are not expressed in either wild-type wing or leg discs, but are enriched in Wg-induced leg discs undergoing transdetermination. Genes in subclusters II and III also had elevated expression in TD cells, but do not have elevated expression levels in wing discs. Subcluster II includes CG12534. Genes that segregated to subcluster IV show high expression in wing discs (yellow) and some of these genes are enriched in TD cells, indicating the realization of a wing developmental program in Wg-induced leg discs. This group includes ap and vg. Genes in subclusters V and VI are expressed at low levels in the TD cell preparation (blue). Components of the Notch signaling pathway and the PcG gene Su(z)2 are included in this group. Gene names for all genes are listed in Tables S7 and S8 (see supplementary material). Some genes are represented by replicate spots on the microarray, causing multiple listings.

View larger version (97K): [in a new window]   Fig. 4. lama expression in larval organs and transdetermined cells. In situ hybridization with a lama probe in young 3rd instar larval organs (A-E), a disc from a wandering 3rd instar (F) and in 1st leg discs from a late 3rd instar (G) induced to transdetermine by ectopic Wg expression. Scale bars: in A, 50 µm for A-E; in F, 50 µm for F,G.

View larger version (17K): [in a new window]   Fig. 5. Involvement of PcG, trxG and lama genes in transdetermination. The frequency of transdetermination was determined (A) by vgBE-lacZ expression and (B) by the occurrence of wing cuticle for control discs, PcG heterozygous mutant discs [Scm, E(z), Pc, E(Pc), and Su(z)2], trxG heterozygous mutant discs (brm and osa) and homozygous lama mutant discs. The frequency of wing cuticle could not be measured for Su(z)2 as no adult survivors were recovered and it was not determined for brm. (C) Determinations of the relative area of transdetermination compared the area of vgBE-lacZ expressing cells with the total disc area (%). For details of the genetic background and t-test statistics, see the legend to Table S11 in the supplementary material.

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