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First published online 18 July 2007
doi: 10.1242/dev.006411

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Recruitment of cells into the Drosophila wing primordium by a feed-forward circuit of vestigial autoregulation

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 (76K): [in this window] [in a new window]   Fig. 1. Wg is not sufficient to activate vg in ap0 wing discs. (A-D) Wild-type Drosophila discs. Vg protein (green in A) is expressed in D-V `border' cells (yellow in A owing to co-expression with Wg, red) as are BE reporter genes (BE-lacZ, blue in C; BE-vgGFP, green in D). Vg is also expressed in surrounding cells of the `pouch' and wing primordium, as are QE reporter genes (5XQE-DsRed, red in B,C; 1XQE-lacZ, blue in D). Wg (red in A,D, green in B) is expressed in border cells within the pouch, and in an inner and outer ring (IR, yellow arrowhead; OR, white arrowhead) in the surrounding hinge primordium. rn-lacZ (blue in B) is expressed throughout the pouch and in the surrounding `rn-only' territory extending up to the inner ring of Wg. (E-H) ap0 discs. Neither Vg (green in E, red in H), nor BE-vgGFP (green in H), nor QE reporters (blue in E,H, red in F,G) are expressed, and the pouch is either absent or present only as a small cluster of rn-only cells (blue in F) surrounded by the inner ring of Wg (red in E, green in F). Uniform ectopic expression of UAS-Nrt-wg under C765-Gal4 control does not rescue expression of either QE reporter gene, endogenous vg expression or wing pouch development (G,H; red stain in G is non-specific tracheal staining). Here, and in the remaining figures, all 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 above each image.

View larger version (65K): [in this window] [in a new window]   Fig. 2. Rescue of Drosophila wing development and vg reporter expression in ap0 discs by UAS-Nintra and UAS-NECN clones. Tub1-Gal4/UAS-Nintra (A) and Tub1-Gal4/UAS-NECN (B-E) clones monitored by autonomous expression of Wg (red in A,C), UAS-GFP (green in B), BE-vgGFP (green in C,D), or BE-lacZ (blue in E), induce expression of both QE reporter genes (1XQE-lacZ, blue in A,C; 5XQE-DsRed, red in B,E), as well as rn-lacZ (blue in B) in surrounding cells (note that rn-lacZ expression extends beyond that of 5XQE-DsRed). Restored growth of the wing pouch and surrounding rn-only territory is indicated by the greatly expanded inner ring of Wg expression (A,C; compare with Fig. 1E,F). Clones induced in early (D) or mid- (E) third instar also induce non-autonomous QE reporter expression, although the range of the response is greater for clones induced during the first instar (A-C). Here, and in the remaining figures, clones were induced during the first larval instar (except for D,E), and the clone genotypes are indicated by colored ovals (representing presence of marker expression within the respective clones), as indicated in each experiment.

View larger version (103K): [in this window] [in a new window]   Fig. 3. Rescue of Drosophila wing development and QE reporter expression by UAS-vg clones. (A-E) Tub1-Gal4/UAS-vg clones [marked by UAS-GFP (green in A,C-E), or the absence of GFP (black in B); clone genotype indicated by colored outline with black filling representing absence of marker expression within the clone] induce Vg (blue in A) and 1XQE-lacZ (red in A,C-E) in surrounding cells in ap0 discs. Tub1-Gal4/UAS-vg clones ectopically express wg (wg-lacZ, blue in B, white arrows) and their ability to induce 1XQE-lacZ in surrounding cells depends on the Wg they secrete. Tub-Gal4/UAS-vg clones that are also wg0 fail to do so (C), unless they co-express exogenous Wg (D), or membrane-tethered Wg (Nrt-Wg, E), in which case they do so at long range, or only in adjacent cells, respectively. Coincident GFP and 1XQE-lacZ expression appear yellow in C,E, and white when co-stained for Wg (blue) in D.

View larger version (88K): [in this window] [in a new window]   Fig. 4. Rescue of wing development and QE reporter expression by Tub1>vg clones depends on Wg input. (A) Tub1>vg clones (black by absence of GFP, green) in ap0 discs express moderate levels of Vg (dull blue) and autonomously rescue Drosophila wing growth when located in the prospective wing pouch; Tub1>vg cells within the rescued pouch also express low levels of the 1XQE reporter (dull red). (B) Tub1>vg clones in ap0; C765-Gal4/UAS-Nrt-wg discs (which overexpress Nrt-Wg throughout the disc) autonomously express normal peak levels of Vg (bright blue) as well as 1XQE-lacZ expression (bright red) within the prospective wing pouch and induce surrounding cells up to several cell diameters away to do the same (coincident GFP and 1XQE-lacZ reporter expression in non-autonomously rescued cells appears yellow).

View larger version (109K): [in this window] [in a new window]   Fig. 5. Tub1>vg clones act at short range to induce vg and QE reporter expression in neighboring UAS-Nrt-wg clones. (A-D) ap0 discs containing Tub1>vg clones (black by absence of DsRed, left column), C765-Gal4/UAS-Nrt-wg clones (yellow by coincident expression of Flu-tagged Nrt-Wg, green, and DsRed), and `double' Tub1>vg C765-Gal4/UAS-Nrt-wg clones (green by expression of Nrt-Wg in the absence of DsRed) are shown stained for Vg (A,C) or 1XQE-lacZ (B,D) expression (blue). (A,B) Double clones (green) within the prospective Drosophila wing pouch (A,B, 2) show peak expression of Vg (bright blue) and 1XQE-lacZ and induce peak expression in adjacent cells outside of the clone (note that the green territories are nested inside the larger bright blue territories, right column). Tub1>vg clones (A, black, 1) show only moderate Vg expression (derived only from Tub1>vg, dull blue). (C,D) Tub1>vg clones (1, black) that abut C765-Gal4/UAS-Nrt-wg clones (3, yellow) induce cells in the latter to express peak levels of Vg and 1XQE-lacZ. The induction is not limited to those C765-Gal4/UAS-Nrt-wg cells that abut the Tub1>vg clone, but propagates over many cell diameters into the C765-Gal4/UAS-Nrt-wg clone. Vg and 1XQE-lacZ expression are also upregulated in the Tub1>vg cells that abut the C765-Gal4/UAS-Nrt-wg clone.

View larger version (104K): [in this window] [in a new window]   Fig. 6. Tub1>vg clones act at short range to induce vg and QE reporter expression in neighboring UAS-arm* clones. ap0 discs containing Tub1>vg clones (#1, black by absence of DsRed) that abut C765-Gal4/UAS-arm* clones (#3, yellow by coincident expression of Flu-tagged Arm* and DsRed) are shown stained for Vg (A) or 1XQE-lacZ (B) expression (blue). Peak levels of both responses propagate throughout the C765-Gal4/UAS-arm* tissue located within the prospective Drosophila wing pouch, but not into neighboring cells outside of the clones (compare with the local non-autonomous induction of both responses by C765-Gal4/UAS-Nrt-wg clones in Fig. 5).

View larger version (116K): [in this window] [in a new window]   Fig. 7. Recruitment of presumptive notal cells to the wing fate by ectopic Wg- and Vg-expressing cells. (A-B') Wild-type Drosophila discs containing Tub1>Gal4/UAS-vg UAS-wg clones located within the presumptive notum, probed for Vg (A, red), 5XQE-DsRed (B, red) and rn-lacZ (B', blue) expression and marked by Vg overexpression (A, bright red) or loss of GFP (B). Arrows indicate clones that have induced `ectopic' wing pouches; note that rn-lacZ expression (B') extends beyond that of 5XQE-DsRed (B), as in the normal pouch. (C-F) Discs that express UAS-vg in A compartment border cells under dpp-Gal4 control [red by ectopic 1XQE-lacZ (D) and Vg (E,F) expression] and contain P compartment clones of Tub1>Nrt-wg cells (indicated by the arrow in C; black by the absence of CD2, green. The arrowed clone in F is shown at higher magnification in E,E'. Cells within the clones that are located within 10-20 cell diameters of the A-P boundary ectopically express normal peak levels of 1XQE-lacZ (D) and endogenous Vg (E') and induce immediately adjacent cells across the clone border to do the same (appear yellow in overlap with CD2). (G-H') UAS>Nrt-wg (G,G') or UAS>arm* (H,H') clones (marked green by Flu epitope staining of Nrt-Wg and Arm*) located within the presumptive notum ectopically express normal peak levels of endogenous Vg (dull red) when they abut UAS>vg clones (bright red by Vg overexpression) and are located within 10-20 cell diameters of the A-P boundary. The UAS>Nrt-wg clones, but not the UAS>arm* clones, also induce their immediate neighbors to do the same.

View larger version (33K): [in this window] [in a new window]   Fig. 8. The feed-forward circuit of Wg-dependent vg autoregulation in Drosophila. DSL-Notch signaling (purple) induces wg (red) and BE-dependent Vg (VgB, green) expression in border cells, causing them to send Wg (red arrows), as well as the short-range feed-forward signal, `X' (black arrows). X entrains neighboring cells to activate QE-dependent Vg expression (VgQ, blue) in response to Wg, and these cells become a new source for X so that the process reiterates, propagating recruitment of surrounding cells into the growing wing primordium (as illustrated on the right). Continuous exposure to X (short black arrows on right), together with Wg, may also be required to sustain QE-dependent vg expression in prospective wing cells once they are recruited. This circuitry is integrated with other signaling processes, including the stimulation of growth of the surrounding cell population (white), from which vg-expressing cells are recruited, to control the expansion in wing size as discussed in the text and the accompanying paper (Zecca and Struhl, 2007).