QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

doi: 10.1242/10.1242/dev.00607

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Janody, F. Articles by Treisman, J. E. Search for Related Content PubMed PubMed Citation Articles by Janody, F. Articles by Treisman, J. E.

Two subunits of the Drosophila mediator complex act together to control cell affinity

Skirball Institute of Biomolecular Medicine and Department of Cell Biology, NYU School of Medicine, New York, NY 10016, USA

View larger version (89K): [in a new window]   Fig. 1. skd- or kto-mutant clones cross the AP compartment boundary. (A-H) Third instar wing discs containing mutant clones, with wild-type tissue labeled with GFP (green; C,D,G,H). Ci, marking anterior cells, is stained red (A,D,E,H). (A-D) skdT606 clone. en-lacZ expression, detected by anti-ß-gal staining (blue), marks the posterior compartment. (E-H) ktoT241 clone. Anti-ß-gal staining (blue) revealing ptc-lacZ expression (F,H). Arrows indicate anterior clones that have crossed into the posterior compartment. (I,J) ktoT663 clone expressing HACi(m1-4), a form of Ci with all the PKA sites mutated. Anti-ß-gal staining (red) reflecting hh-lacZ expression; the clone is positively marked with GFP (J). Ci activation does not rescue the boundary crossing behavior. The compartment boundary is indicated by a white dashed line (D,H,J). (K) skd- and kto-mutant clones are rounder than their wild-type twin spots, and skd kto double-mutant clones are equally round. Circularity is measured as 4A/L2, where A is the area of the clone and L is the perimeter, and would be 1.0 for a perfect circle. The mean for wild-type twin spots is 0.41, for skd clones is 0.67, for kto clones is 0.65 and for skd kto clones is 0.74. Lines within the bars indicate±1 s.d. P<0.001 for a comparison of skd, kto or skd kto to wild type. The differences between skd, kto and skd kto are not significant.

View larger version (102K): [in a new window]   Fig. 2. skd- or kto-mutant clones distort the DV compartment boundary. (A-H) Third instar wing discs containing skdT606-mutant clones. Wild-type tissue is labeled with GFP (green; C,D,G,H). ap-lacZ expression, marking the dorsal compartment, is stained with anti-ß-gal (blue; A,D,E,H). Wg protein is stained red (B,D,F,H). Arrows indicate clones that distort the DV boundary, producing a bulge in the Wg stripe and a distortion of the ap expression boundary. (A-D) A clone with dorsal cells in the ventral compartment. (E-H) A clone with ventral cells in the dorsal compartment. (I-J) A ktoT631-clone entirely within the ventral compartment that has distorted the DV boundary (arrow). Wild-type tissue is labeled with GFP (green; J) and stained with anti-ß-gal (red) to show ap-lacZ expression (I,J). (K,L) Second instar wing discs containing skdT413-mutant clones. Wild-type tissue is labeled with GFP (green; L). Anti-ß-gal (red) staining reflecting caps-lacZ expression (K,L). Arrows indicate dorsal clones that continue to express caps-lacZ. Note that the caps-lacZ transgene is on the same chromosome arm as skd and thus is not present in the wild-type twin spots (asterisks in K and brighter green staining in L).

View larger version (89K): [in a new window]   Fig. 3. skd kto double mutants have the same phenotype as either single mutant. (A-F) Third instar eye discs. Wild-type tissue is labeled with X-gal staining, revealing arm-lacZ expression (A-D), or with GFP (green; F). (A-C) Elav-stained photoreceptors (brown). (A) skdT616 clones, (B) ktoT241 clones, and (C) skdT606, ktoT241 clones. Few Elav-stained photoreceptors form at the posterior of all clones, although cell growth and survival are unaffected. (D-F) Atonal staining of ktoT314 clones (brown; D) or skdT606, ktoT241 clones (red; E,F). Ato is inappropriately maintained posterior to its normal domain in single- or double-mutant clones. (G-I) Third instar wing discs with skdT606, ktoT241 clones. Wild-type tissue is labeled with GFP (green; I). Ci is stained red (G,I) and Wg blue (H,I). Double-mutant clones cross the AP compartment boundary (white dashed line in I) and distort the DV boundary (arrow; H). Ci and Wg are expressed at normal levels within the mutant tissue.

View larger version (79K): [in a new window]   Fig. 4. skd and kto act synergistically and influence cell affinity when overexpressed. (A-C) Adult wings from flies overexpressing two copies of skd (A), two copies of kto (B), or one copy of skd and one copy of kto (C) with vg-GAL4. The wing margin is reduced by overexpression of either gene alone, but the effect is much stronger when both are co-expressed. (D-I) Wing discs, stained with Wg (red), in which clones expressing two copies of skd (D,G) two copies of kto (E,H), or one copy of skd and one copy of kto (F,I) have been induced. All clones are positively labeled by GFP expression (green; G-I). Arrows indicate clones that distort the DV boundary, producing a bulge in the Wg stripe. (J) Clones overexpressing two copies of kto, or one copy each of skd and kto, are rounder than clones overexpressing two copies of skd. Mean circularity for two copies of skd is 0.44, for two copies of kto 0.78 and for one copy of skd and one copy of kto 0.71. Lines within the bars indicate ±1 s.d. P<0.001 for a comparison of two copies of skd to skd and kto. The difference between two copies of kto, and skd and kto is not significant. (K) Size distribution of clones overexpressing two copies of skd (yellow), two copies of kto (orange), or one copy of skd and one copy of kto (green). Clones overexpressing both skd and kto are smaller in size. A total of 173 clones overexpressing two copies of skd, 66 clones overexpressing two copies of kto and 186 clones overexpressing both genes were analyzed.

View larger version (54K): [in a new window]   Fig. 5. The Skd and Kto proteins associate in vivo. (A-F) Third instar eye discs. (A-C) show skdT413-mutant clones. Wild-type tissue is labeled by anti-ß-gal staining reflecting arm-lacZ expression (green; B,C). Rabbit anti-Skd staining (red; A,C). (D-F) ktoT241-mutant clones. Wild-type tissue is labeled with GFP (green; E,F). Rat anti-Kto staining (red; D,F). In both cases, the antibodies do not stain mutant tissue, but show ubiquitous nuclear staining in wild-type tissue. The same pattern was observed for the guinea pig anti-Kto antibody (not shown). (G-H) Coimmunoprecipitation of Skd, Kto and SOH1. (G) Coimmunoprecipitation of Kto and Soh1 with an anti-Skd antibody. The upper gel is blotted with rat anti-Kto and the lower gel with anti-SOH1. Lane 1, 10% white- total embryonic extract; lane 2, IP with anti-Skd from white- extract; lane 3, control IP from white- extract; lane 4, IP with anti-Skd from extract overexpressing UAS-skd and UAS-kto with da-GAL4; lane 5, control IP from extract overexpressing UAS-skd and UAS-kto with da-GAL4. (H) Coimmunoprecipitation of Skd and Soh1 with the guinea pig anti-Kto antibody. The upper gel is blotted with anti-Skd and the lower gel with anti-SOH1. Lane 1, 2.5% white- total extract; lane 2, IP with anti-Kto from white- extract; lane 3, control IP from white- extract; lane 4, IP with anti-Kto from extract overexpressing UAS-skd and UAS-kto with da-GAL4; lane 5, control IP from extract overexpressing UAS-skd and UAS-kto with da-GAL4.