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First published online October 27, 2004
doi: 10.1242/10.1242/dev.01450

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Differing strategies for the establishment and maintenance of teashirt and homothorax repression in the Drosophila wing

¹ Department of Biological Sciences, Columbia University, 701 West 168th Street, HHSC 1104, New York, NY 10032, USA
² Department of Biochemistry and Molecular Biophysics, Columbia University, 701 West 168th Street, HHSC 1104, New York, NY 10032, USA

View larger version (67K): [in a new window]   Fig. 1. Subdivision of the proximodistal (PD) axis in the wing disc. (A) Cartoon of a third instar wing imaginal disc, showing wing pouch (P), distal hinge (DH), proximal hinge (PH), lateral hinge (L) and notum (N). Anterior is up and dorsal is to the left in all figures. (B) Adult wild-type wing showing PD subdivisions. Labels are the same as in A, except for the wing blade (WB), which fate maps to the pouch. (C-E) Late third instar wing disc stained for Tsh (red) and Hth (blue). Neither is expressed in the pouch. Hth extends throughout the entire hinge, while Tsh is restricted to the PH. A deep fold in the epithelium (yellow arrowhead in C) marks the separation between DH and PH.

View larger version (112K): [in a new window]   Fig. 2. Repression of hth, but not tsh, requires Dpp signaling. (A-D) Adult wings containing multiple Medadro clones induced in a Minute background by vgBE::flp. Mutant clones are marked by y- tissue, and denoted by black arrows. (A) Medadro clones result in reduced growth of the wing blade and hinge along the PD axis. (B) A mutant Medadro clone sorts out from surrounding wild-type tissue in the wing blade. (C) A mutant Medadro clone differentiates wing margin bristles and non-autonomously induces wing margin elements in adjacent wild-type cells. (D) Medadro clones located near the anteroposterior (AP) compartment boundary of the hinge differentiate bristles characteristic of the lateral hinge. (E-U) Immunostains of wing discs; Hth staining is in blue, Tsh is in red, and clones are marked by the absence of CD2 in green. (E-H) Late third instar wing disc containing Medadro M+ clones induced by vgBE::flp. (F-H) Magnification of the region denoted by the white box in E. A large mutant clone in the wing pouch ectopically expresses hth, but not tsh. (I-K) Late third instar wing disc containing Medadro M+ clones induced by heat shock in the mid-third instar. (J,K) Magnification of the region denoted by the white box in I. The yellow arrowheads point to ectopic hth expression in small wing pouch clones. (L) Mid third instar wing disc containing flip-out clones of brk marked by GFP, and stained for Hth and Tsh. The magnification shows a clone that fails to repress hth but has no effect on tsh expression. (M) Mid third instar wing disc containing flip-out clones of brk marked by GFP, and stained for Nubbin (Nub) and Tsh. The magnification shows a clone that has no effect on either tsh or nub expression. (N-Q) Late third instar wing disc containing Medadro M+ clones induced by heat shock in the first instar. tsh repression is maintained in distal cells, which strongly express hth. (R-U) Late third instar wing disc containing Medadro M+ clones induced by heat shock in the first instar. The distal limit of tsh repression coincides with the proximal limit of Dll (violet) expression.

View larger version (135K): [in a new window]   Fig. 3. Repression of hth near the AP boundary does not require vg. (A-C) Wild-type third instar wing imaginal disc, stained for Hth (blue) and Vg (red). (A) White arrows point to the lateral hinge, where hth and vg are co-expressed at high levels. (D-I) Clones are labeled by the loss of Vg protein (red), and the loss of vgQE-lacZ and arm-lacZ expression (green). (D-F) Late third instar wing disc containing multiple vg– M+ clones, induced in the early third instar. Hth (blue) is not de-repressed in wing pouch clones near the AP boundary (a subset are outlined in white), but clones in the lateral wing pouch express Hth (yellow arrowheads). (G-I) Late third instar wing disc containing a large vg– M+ clone (white outline) induced in the second instar. The clone expresses two rings of Hth (blue), separated by a large ectopic fold.

View larger version (75K): [in a new window]   Fig. 4. Antagonism between Vg and Hth requires Dpp signaling. In all panels, Hth staining is in blue, Vg is in red, and clones are marked by the absence of GFP or CD2 in green. (A-D) vgBE::flp-induced Medadro M+ clone in the wing pouch. (B-D) Magnification of the region denoted by the white box in A. hth is de-repressed even though vg is still highly expressed in the clone (white outline). (E-H) brk– clones, induced by heat shock during the second instar. The yellow arrowhead points to a lateral DH clone, which loses hth expression and maintains vg expression. The white dotted line marks the epithelial fold between the DH and PH.

View larger version (120K): [in a new window]   Fig. 5. tsh repression is not maintained by Wg or Dpp signaling. In all panels, Hth staining is in blue and Vg is in red. (A-C) Late third instar wing disc containing arr– clones, induced by heat shock during the second instar. Clones are marked by the absence of GFP. hth is only de-repressed in arr mutant clones away from the AP boundary, while tsh is never de-repressed in the pouch (yellow arrowheads). (D-F) dTCFDN expression in clones (GFP positive), induced by heat shock during the second instar. Clones far from the AP boundary in the pouch de-repress hth, but have no effect on tsh expression (yellow arrowheads). Note that in the hinge, both arr– and dTCFDN clones ectopically express tsh (A-F, white arrows), indicating a failure to maintain tsh repression in this domain. (G-K) dTCFDN expression (GFP positive) in Med13 M+ clones induced in the second instar (8 clones scored). (G-I) hth, but not tsh, is ectopically expressed in mutant pouch cells (yellow arrowheads). (J) tsh is still repressed in a pouch clone (white outline) that no longer stains for Dll (violet), a marker of Wg signal transduction. (K) In the peripodial membrane the endogenous tsh and hth expression is unaffected by a large clone (white outline).

View larger version (121K): [in a new window]   Fig. 6. PcG genes are necessary for tsh, but not hth, repression. (A-C) Adult wing containing multiple Su(z)12daed clones induced by vgBE::flp. Mutant clones are marked by y– tissue, and are denoted by black arrows. (A) Note the reduction of growth along the PD axis. (B) A Su(z)12daed clone sorts out from surrounding wild-type wing tissue. (C) A Su(z)12daed clone differentiates bristles characteristic of more proximal cuticle. (D-M) Immunostaining of wing discs with Su(z)12daed or Pc– clones. Hth staining is in blue and Tsh is in red. Mutant tissue is marked by the absence of GFP. (D-F) vgBE::flp-induced Su(z)12daed clones in the wing pouch. tsh is ectopically expressed at high levels throughout much of the mutant tissue. hth is weakly expressed in a subset of tsh-expressing cells. (G-I) Like Su(z)12daed clones, vgBE::flp-induced Pc– clones de-repress tsh but not hth. (J-M) Expression of the distal wing fate markers Dll (J,K) and nub (L,M) is lost or reduced in Su(z)12daed mutant tissue (yellow arrowheads).

View larger version (118K): [in a new window]   Fig. 7. Temporal and spatial regulation of tsh repression by Pc. (A) Time line of clone induction. Red bars indicate windows in which Pc- clones were induced by heat shock relative to hours after egg laying (AEL) and tsh repression. Note that the 72-84 and 84-96 hours AEL time points occur after the initiation of tsh repression in the second instar. (B-M) tsh expression (red) in Pc– clones, marked by the absence of GFP. (B,C) Pc– clones induced 84-96 hours AEL express tsh in the most distal cells of the hinge (white arrow), but not in the wing pouch. (D,E) Pc– clones induced 72-84 hours AEL express tsh in the wing pouch and distal hinge (white box, enlarged). (F,G) tsh expression levels in Pc– clones induced 48-60 hours AEL are comparable to endogenous hinge and notum levels. (H,I) Large Pc– clones induced 24-36 hours AEL. Although tsh is widely expressed in the mutant tissue, it is not expressed in cells close to the DV boundary (white box, enlarged) or PH (yellow arrowhead). (J-M) Pc– MARCM clones, marked by GFP, induced in early second instar larvae. (J,K) Control Pc– clones de-repress tsh. (L,M) Pc– clones expressing Nrt-Wg do not de-repress tsh.

View larger version (58K): [in a new window]   Fig. 8. Model of tsh and hth regulation in the wing disc. In all panels: P, pouch; DH, distal hinge; PH, proximal hinge; N, notum; green, Vg; blue, Hth; pink, Tsh+Hth. (A) Early in the second instar, prior to the formation of the DV compartment boundary, the ventro-anterior wedge of Wg represses tsh in the prospective appendage (1). Following this establishment period, PcG members maintain tsh repression independently of Wg and Dpp signaling (2), although tsh remains sensitive to Wg-mediated repression. (B) Late in the second instar, after the formation of the DV boundary, Dpp (yellow arrows) represses hth in cells that have upregulated vg. (C) Third instar wing disc, showing the complex relationship between Vg, Hth, and the Dpp gradient. Unlike tsh, hth repression requires continuous signal input. In the medial region of the pouch, high levels of Dpp completely repress Brk, allowing the expression of a putative hth repressor (R), irrespective of Vg. In the lateral regions of the pouch, moderate Dpp levels incompletely repress Brk, and Vg is required to ensure sufficient levels of R to maintain hth repression. In the lateral DH, low levels of Dpp signaling fail to repress Brk, which represses R, permitting the co-expression of Vg and Hth. Note that the above model does not apply to the PH, where hth is insensitive to repression by Dpp signaling, possibly due to the presence of Tsh.