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Repression of Teashirt marks the initiation of wing development

Jun Wu^* and Stephen M. Cohen

Developmental Biology program, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany
^* Present address: Mount Sinai School of Medicine, 1 Gustav Levy Place, New York, NY, USA

View larger version (76K): [in a new window]   Fig. 1. Early expression of Tsh, Hth, Vestigial, Wg and Nubbin. (A) Expression of Vestigial (blue), Hth (red) and Tsh (green) proteins in an early second instar wing disc. The overlay is show on the left. Single channels are shown on the right. Tsh expression is low in the area indicated by the arrow. The white line indicates the position of the optical cross section shown in B. Cells expressing low levels of Tsh appear purple in the overlay on the left. The diagram on the right indicates the orientation of the optical cross sections. (C) Expression of Vestigial, Hth and Tsh in a mid second instar wing disc. Tsh is repressed in presumptive wing (arrow). The white line indicates the position of the optical cross section shown in D. (E) Expression of Tsh, Nubbin (blue) and wg-lacZ (anti-ß-gal, red) in a mid second wing disc. Tsh is repressed in presumptive wing (arrow) where wg-lacZ is expressed. Nubbin expression is not detected at this stage (white in single channel). (F) Cross-section of E.

View larger version (86K): [in a new window]   Fig. 2. Expression of Tsh, Hth, Vestigial, Wg and Nubbin at later stages of wing development. (A) Tsh protein (green) is repressed in wg-lacZ expressing cells (anti-ß-gal, red) in a late second instar disc. Nubbin is faintly detectable (blue in overlay, white in single channel image). (B) Hth (red), Vestigial (green) and Nubbin (blue/white) expression in an early third instar disc. Hth begins to be repressed in the wing pouch. Nubbin is expressed in wing pouch cells. (C) Cross-section of B. Hth is still expressed in some Vestigial-expressing cells (arrow), suggesting that Hth is repressed after Vestigial is expressed. (D) Nubbin, Hth and Tsh expression in a late third instar wing disc. The magnification of D is 50% that of the other panels. The spatial relationships in the proximal regions are better illustrated in the cross-section shown in E. Nubbin is expressed in the wing pouch. Hth expression at this stage is different from continuous expression pattern at earlier stages shown in B,C. Hth is expressed in three separate domains as labeled as 1, 2, 3. Nubbin expression overlaps with Hth domain 1 and 2. Tsh overlaps partly with Hth domain 3 (yellow, arrow). Tsh is expressed proximal to Hth ring 3. Tsh and Hth are co-expressed in the notum. (F) Cross-section of a late third instar wing disc showing Hth, Vestigial (blue) and Wg (green). Vestigial is expressed in the wing pouch. The proximal rings of Wg expression overlap with Hth domain 2 and 3. (G) Cross of a mid-late third instar disc. The wg-lacZ rings (anti-ß-gal, red) are distal to Tsh expression. 2, 3 denote the positions of Hth rings 2 and 3, which overlap the two rings of wg expression. Nubbin overlaps Hth ring 2 and the first wg ring.

View larger version (51K): [in a new window]   Fig. 3. Tsh is repressed normally in vestigial, apterous and nubbin mutant wings. (A) Tsh expression in an apUG035 mutant wing disc. (B) Tsh expression in a vg83b27r mutant wing disc. (C) Tsh expression in a nub1 mutant wing disc.

View larger version (104K): [in a new window]   Fig. 4. Tsh and Hth expression in genetic mosaics. (A) vg83b27r mutant clones were induced between 48 and 72 hours after egg laying. Early third instar larvae were examined 2 days after clone induction, so that the majority of clones examined were induced in early second instar. Clones were labeled by absence of ß-gal (blue). Hth (red) was ectopically expressed in the clone. Tsh (green) was not. Higher magnification views of the boxed region are shown separately for each channel at right. 11 of 13 clones examined expressed ectopic Hth, but Not Tsh. The relatively large size of the clone shown here may reflect some growth prior to wing pouch specification. (B) Clones expressing Vestigial (Act>Gal4 UAS-Vg UAS-GFP) induced during second instar and examined in mature third instar discs. Vestigial-expressing clones marked by co-expression of GFP (blue). Hth (red) levels were reduced in the mutant cells. Tsh (green) levels were unaffected relative to adjacent wild-type cells. Note that the Vg-expressing clones tend to sort out from the disc (arrowhead) and so the nuclei of the cells are not uniformly in the same focal plane. (C) nub1 mutant clones induced in early third instar 72-96 hours after egg laying. Clones were labeled by absence of ß-gal. Hth was ectopically expressed in a nub1 clone whereas Tsh was not (arrow).

View larger version (88K): [in a new window]   Fig. 5. Tsh is not repressed in wg mutant discs in second instar. (A,B) Second instar wg1/wg17en40lacZ wing disc labeled to visualize Tsh (green), Vestigial (red) and wg-lacZ (blue). (B) An optical section perpendicular to the lane of the section in A. (C) Third instar disc of the same genotype labeled to visualize Wg (blue) and Tsh (red). Note the uniform expression of Tsh and the symmetric duplication of the characteristic notum stripe of Wg expression.

View larger version (149K): [in a new window]   Fig. 6. Tsh is repressed by Wg signaling. arms10-expressing clones were induced in y w act>CD2>Gal4/y w HSflpI; UAS-armS10 /UAS-GFP larvae 24-48 hours after egg laying (A), 48-72 hours after egg laying (B,C) and 72-96 hours after egg laying (D). arms10-expressing clones were labeled by co-expression of GFP (red). (A) Optical section of an early third instar wing disc containing a clone induced in first instar. Tsh (green) was repressed autonomously in the clone. Hth (blue) was not. Tsh and Hth channels shown separately at right. (B) Optical section of a mid third instar wing disc containing multiple arms10-expressing clones. Higher magnification views of the boxed region are shown on the right. Tsh (green) was repressed in arms10-expressing clones (e.g. arrows). Hth (blue, or white in single channel) expression was not reduced in these clones. In some clones, Hth was upregulated. (C) Optical section of a late third instar wing disc containing multiple arms10-expressing clones labeled with anti-Tsh and anti-Nubbin (blue, or white in single channel). Most clones repressed Tsh but did not express Nubbin (e.g. arrow). Some clones expressed low levels of Nubbin (arrowhead). (D) Optical section of a late third instar wing disc containing multiple arms10-expressing clones labeled with anti-Tsh and anti-Vestigial (blue, or white in single channel). Vestigial was expressed in most clones although not in every cell of the clone (arrowheads). Some clones do not express Vestigial (arrow). Tsh was repressed in cells lacking ectopic Vestigial expression.

View larger version (53K): [in a new window]   Fig. 7. Tsh is repressed by Dpp signaling. Clones expressing activated Thickveins were induced in larvae of the following genotype: y w act>CD2>Gal4/y w HSflpI; UAS-tkv* /UAS-GFP. Clones were induced at 48-72 hours after egg laying. An optical section of a late third instar wing disc is shown. Tsh (green) expression is repressed in tkv*-expressing clones (arrows). Hth (blue, or white in single channel) is repressed in some cells of the lateral clones. Hth is not repressed by clones in the Hth ring 3 (arrowhead). Cells that express both proteins appear pink.

View larger version (75K): [in a new window]   Fig. 8. Repression of Tsh is required for wing pouch formation. (A) scallopedGal4 UAS-Tsh wing disc labeled to visualize Wg (red), Hth (blue) and Tsh (green). Note the absence of the wing pouch and the lack of the DV boundary stripe and rings of Wg expression. The folding of the discs makes it difficult to get all nuclei in the same horizontal optical section. The optical cross section at right shows that Hth was expressed in all nuclei, indicating that wing pouch specification has been blocked (B) dppGal4 UAS-Tsh wing disc labeled to visualize Vg (red), Nub (blue) and Tsh (green). Nub was partially repressed in the dorsal and ventral compartments. We also noted stronger repression in a part of the dorsal territory. The basis for this difference is not clear, but we noted weak Tsh expression in much of the stronger repression domain. Perhaps this reflects intrusion of tissue from the hinge region due to ectopic Tsh expression.

View larger version (13K): [in a new window]   Fig. 9. The three major stages of wing development: (1) repression of Tsh by Wg and Dpp signaling; (2) Loss of Tsh allows Notch-dependent activation of Wg and Vestigial (Vg) boundary enhancer expression at the DV boundary; and (3) long-range Wg gradient induces several genes required for wing growth and patterning, e.g. Vg.