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First published online 18 July 2007
doi: 10.1242/dev.006445

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Control of Drosophila wing growth by the vestigial quadrant enhancer

Howard Hughes Medical Institute, Department of Genetics and Development, Columbia University College of Physicians and Surgeons, 701 W 168th Street, New York, NY 10032, USA.

View larger version (108K): [in this window] [in a new window]   Fig. 1. Vg expression, QE activity and wing development in wild-type, vgb and vg0 wing discs. (A,A') 1XQE-lacZ (A, ß-gal, red) and Vg (A', blue) expression define the wing primordium of the mature Drosophila wing disc (also delimited by a characteristic fold, white dots). The surrounding rotund (rn)-only domain (unlabeled) is circumscribed by the inner of two rings of Wg expression [green; inner ring (IR), yellow arrowhead and yellow dots in A-C,E; outer ring (OR), purple arrowhead]. (B,C) Vg and 1XQE-lacZ expression and the wing primordium are absent, and the rn-only domain is reduced or eliminated in vgb (B) and vg0 (C) discs. (D-E') Vg and 1XQE-lacZ expression and wing development are rescued in rp49-vg vgb discs (D,D'), but not in rp49-vg vg0 discs (E,E'; except for a few patches of weak 1XQE-lacZ expression visible in the overexposed image in E'). The rn-only domain appears rescued in both rp49-vg vgb and rp49-vg vg0 discs. Vg expression is also restored in D-V border cells of rp49-vg vgb discs (albeit only in the wing pouch), indicating additional BE(s) in the vgb gene. (F-G') Tub1>vg clones (black by the absence of GFP, green) autonomously rescue 1XQE-lacZ expression and wing development in vgb (F,F') and vg0 (G,G') discs. 1XQE-lacZ is expressed in a quadrant pattern in both cases, presumably in response to Wg-expressing (faint green) D-V border cells. Vg expression (bright blue) appears normal in the wing pouch of the vgb disc (F'), indicating a normal QE response by the vgb allele superimposed on the uniform, moderate level of exogenous Vg (dull blue, compare F',G'). Here, and in the remaining figures, clones were induced during the first larval instar (unless otherwise stated), discs are from mature third instar larvae, anterior is left, dorsal is up, protein or reporter gene stains are indicated by color, and relevant genotypes are indicated either above the images or, in the case of clones, indicated by outlined ovals filled in red, blue or black, as marked in the experiment.

View larger version (84K): [in this window] [in a new window]   Fig. 2. Vg expression and wing development in vgb clones; evidence that QE activity requires `priming' by Vg. (A-C,E,E') vgb (A,B,E,E') and vg0 (C) clones (black by the absence of GFP, green) induced before (A,E,E') or after (B,C) D-V segregation. Both vgb and vg0 clones contribute normally to the proximal hinge and notum primordia. However, vgb clones induced before the D-V segregation often fail to contribute to the wing pouch (A,F) or to express 1XQE-lacZ (E', red), whereas clones induced afterwards succeed (B, inset; see F). vg0 clones invariably fail to contribute to the wing pouch (C). For A-D, the sibling `twin' clones are marked by doubled GFP expression, bright green. For E, the Minute technique was used to give the vgb clone a growth advantage. Numbers correspond to time of clone induction in hours after egg laying (h AEL); the D-V segregation occurs at 60 h AEL. (D) Early-induced vgb clones generated in homozygous rp49-vg discs contribute to the wing pouch and express 1XQE-lacZ (red). (F) Bar charts showing the survival of vgb or control (vg+) clones in the wing pouch, relative to that of their wild-type twin clones, depending on the time of induction (h AEL) and the absence or presence of rp49-vg. Bars represent the percentage of wild-type twin clones that contribute to the pouch (1) with an associated vgb clone that contributes to the pouch (green), (2) without an associated vgb clone (red), or (3) with an associated vgb clone that contributes only to the hinge primordium (yellow). n, total number of clones scored for each experimental condition. In the absence of the rp49-vg transgene, early-induced vgb clones contribute only rarely to the pouch, and appear instead to sort into the hinge primordium or to be lost. The ratio of vgb clones that contribute to the pouch (type 1, green) increases at the expense of the other two types as a function of time of induction, reaching the wild-type distribution after the D-V segregation. In rp49-vg discs, both early- and late-induced vgb clones contribute almost normally to the pouch.

View larger version (100K): [in this window] [in a new window]   Fig. 3. 5XQE transgene expression requires Wg input. (A) 5XQE>CD2>vg expression (CD2, red) in a wild-type wing disc (the D-V boundary is marked by BE-vgGFP expression, green). (B,C) Rescue of wing development in vg0 5XQE>vg clones [black by the absence of both GFP and CD2, and blue by the expression of 1XQE-lacZ (B) or Vg (C); vg+ 5XQE>CD2>vg tissue appears yellow]. Note in C that the level of Vg expressed within the vg0 5XQE>vg clone (outlined in green) is similar to that in neighboring wild-type tissue (vg+ 5XQE>CD2>vg, outlined in red). (D) Small, late-induced arr0 clones (black by absence of DsRed, green; white arrows) in a 5XQE>CD2>vg wing disc composed almost entirely of large, early-induced 5XQE>vg clones (remaining patches of arr+ 5XQE>CD2>vg-expressing tissue are marked by CD2 expression, red). 5XQE>vg (Vg, blue) is reduced or absent in the arr0 clones. (E) 5XQE-DsRed expression (blue) is lost in an arr0 clone located inside a Tub1>vg clone (black arrow; black by the absence of both CD2, green, and GFP, red), and in arr0 clones in the surrounding Tub1>GFP>vg tissue (white arrows; appears red).

View larger version (75K): [in this window] [in a new window]   Fig. 4. 5XQE transgene expression requires the vg-dependent feed-forward signal. (A) vg0 disc carrying two types of clones: Tub1>vg clones, black by the absence of GFP (red) and 5XQE>vg clones, black within the prospective Drosophila wing pouch (outlined by dotted line) by the absence of CD2 expression (green). Here (and in B,C), expression of the 5XQE>vg transgene is monitored indirectly in Tub1>GFP>vg tissue by robust expression of 1XQE-lacZ (appears lavender). As illustrated at the bottom, the Tub1>vg clone (green) has induced adjacent cells in the abutting 5XQE>vg clone (lavender) to express the 5XQE>vg transgene, and induction of the 5XQE>vg transgene has propagated through the clone and induced 5XQE>CD2>vg expression (yellow) in neighboring cells on the other side. Expression of both the 1XQE-lacZ and 5XQE>CD2>vg transgenes are rescued within the Tub1>vg clone (as in Fig. 1F,G). (B,C) vg0 discs that carry abutting Tub1>vg and 5XQE>vg clones, as in A, except that the 5XQE>vg and Tub1>vg clones are black by, respectively, absence of GFP (green, owing to excision of a >Tub1-GFP> Flp-out cassette) and absence of DsRed (red). As in A, the Tub1>vg clones in both discs (green in the diagram) have induced 5XQE>vg expression (lavender) that propagates into the abutting 5XQE>vg clones, rescuing wing development.

View larger version (128K): [in this window] [in a new window]   Fig. 5. Control of Drosophila wing growth mediated by the 5XQE element. (A-D) vg0 BE-vgGFP 1XQE-lacZ discs, either lacking (A) or carrying the 5XQE>CD2>vg (B,C) or 5XQE>Tub1-GFP>vg (D) transgene, and either lacking (A,B) or bearing (C,D) early-induced 5XQE>vg clones. 5XQE>vg clones derived from the 5XQE>CD2>vg transgene (C) are black within the prospective wing pouch by absence of CD2 (red) coupled with 1XQE-lacZ expression (blue), which is strongly upregulated in cells in which the 5XQE>vg transgene is active. 5XQE>vg clones derived from the 5XQE>Tub1-GFP>vg transgene (D) are black by the absence of GFP (green); they express 1XQE-lacZ, as in C, when located within the rescued wing pouch. BE-vgGFP expression (green) is only barely and sporadically detectable along the D-V boundary of vg0 BE-vgGFP 1XQE-lacZ discs (A), typically yielding small, anterior and posterior patches of wing tissue, encircled by rings of Wg expression (purple) in the hinge primordium. Addition of the intact 5XQE>CD2>vg transgene (B) yields detectably stronger BE-vgGFP expression and is associated with weak, local expression of 1XQE-lacZ, possibly owing to the contribution of cryptic, low-level Vg derived from the added transgene. 5XQE>vg clones generated in this background (C,D) show significant rescue of wing growth, as visualized by the expanded domains of 1XQE-lacZ-expressing cells. They also induce neighboring cells outside the clone to activate the 5XQE>CD2>vg transgene (red). (E-H) Same as in A-D, except for the added presence of one copy of the rp49-vg transgene, which largely rescues BE-vgGFP expression along the D-V boundary (E) and augments the weak, local activity of both the 1XQE-lacZ and 5XQE>CD2>vg transgenes in the absence of 5XQE>vg clones (E,F, compare with A,B). 5XQE>vg clones in this background cause dramatic expansions in wing growth (G,H), approximating to or exceeding that normally observed in wild-type discs, and are associated with local induction of the intact 5XQE>CD2>vg transgene in neighboring cells outside the clone (arrows). Note that 1XQE-lacZ expression is confined to the prospective wing pouch, demarcated by the inner ring of Wg (purple, arrow in H), even though the clone extends into the proximal hinge territory, indicating an independent limit to the propagation of 5XQE>vg expression.

View larger version (97K): [in this window] [in a new window]   Fig. 6. Enhanced potency of the QE response increases wing growth. (A-D) 5XQE>vg clones in wild-type Drosophila discs, black by the absence of CD2 (green, A,D), and by robust expression of 1XQE-lacZ (red, A,B) or Vg (C, blue). The clones are associated with expansion of the 1XQE-lacZ-expressing (wing) tissue, apparently at the expense of the rn-only domain, normally circumscribed by an inner and outer fold (dotted white and yellow lines, B-D, as indicated by the arrows). The disc in B is counterstained with Wg (blue) and uniform expression of Hsp70-GFP (green) to visualize the folds; the outer fold correlates with the inner ring of Wg expression. (D) Dll expression (summed staining for Dll and a Dll-lacZ transgene, blue) is not expanded within such 5XQE>vg clones. (E) Same as in D, except for the added presence of ubiquitous, ectopic Wg, driven under C765-Gal4/UAS control. Dll expression now extends into the expanded domain of wing tissue (marked by 5XQE-DsRed expression, red).

View larger version (41K): [in this window] [in a new window]   Fig. 7. Model for the control of Drosophila wing growth by feed-forward autoregulation of vg mediated by the QE and fueled by Wg. During the first larval instar (L1), tsh and vg are coexpressed in the nascent wing disc, the latter driven at least in part by the vg priming enhancer (PE). Wg signaling in early L2 represses tsh in the distal portion of the disc, segregating the disc into heritably distinct hinge/notum and pre-blade primordia. Both primordia are then subdivided into dorsal (D) and ventral (V) compartments in mid-L2. After the D-V segregation, vg expression becomes dependent on DSL-Notch signaling (orange) across the D-V boundary, which activates both wg and vg expression in D-V border cells, the latter driven by vg boundary enhancer (BE) elements. By early L3, Wg (blue) and the Vg-dependent feed-forward signal (yellow) sent by border cells activate vg quadrant enhancer (QE) elements in neighboring cells, upregulating vg expression (red). During the mid- and late L3, the domain of vg-expressing cells expands dramatically by reiterative cycles of short-range feed-forward signaling, fueled by long-range Wg and Dpp signaling from D-V border and A-P border cells (for simplicity, Dpp and the A-P compartment boundary are not shown). Feed-forward autoregulation is required both to recruit new cells to express vg, as well as to maintain vg expression in cells already recruited; Wg and Dpp are also required for the survival and proliferation of cells already recruited. Lastly, Vg-expressing cells produce a signal (green) that stimulates proliferation in the surrounding, `rn-only' cells, sustaining the cell population from which wing cells are recruited. Short-range (DSL-N, Feed-forward and Growth) and long-range (Wg) signals are indicated, respectively, by arrowheads and arrows.