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First published online 28 January 2009
doi: 10.1242/dev.032854

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SoxF is part of a novel negative-feedback loop in the wingless pathway that controls proliferation in the Drosophila wing disc

¹ Centro Andaluz de Biología del Desarrollo (CABD), CSIC-Universidad Pablo de Olavide, Sevilla 41013, Spain.
² IPATIMUP, Universidade do Porto, Porto 4200-465, Portugal.
³ IBMC, Universidade do Porto, Porto 4150-180, Portugal.

View larger version (89K): [in this window] [in a new window]   Fig. 1. SoxF is expressed in the hinge abutting the wg IR and rn domains. (A,B) Wild-type wing imaginal disc of a late L3 Drosophila larva counterstained with the actin marker rhodamine-phalloidin (B is a cross-section of the disc shown in A). The prospective hinge is formed by two concentric folds of the disc's epithelium that surround the wing pouch (pink). These two folds, called the distal (DH) and proximal (PH) hinge, give rise to the adult distal and proximal hinge structures, respectively, that articulate the wing blade (distal) (false-colored pink) with the thorax (body wall, false-colored yellow). The wg outer (OR) and inner (IR) rings are marked by an arrow and an arrowhead, respectively, throughout the figure, and are false-colored as red stripes in A-C. (C) Adult structures derived from the wing disc: wing blade (pink), notum (yellow; only a portion of this structure is shown) and hinge. The position of the two wg expression stripes in the hinge is marked with an arrow (IR) or arrowhead (OR). (D) Late L3 wing disc of MS209-GAL4;UAS-GFP. This line drives GFP expression specifically in hinge cells. The disc is counterstained with rhodamine-phalloidin. (E) rn-lacZ (rnZ) disc stained for β-galactosidase, Wg protein and SoxF transcription (fluorescent in situ hybridization). (F) High-magnification view of the dorsal hinge region of a rn-lacZ wing disc also stained for Wg antigen. The expression of wg in the DH (IR; arrowhead) lies at the border of the rn-lacZ domain. (G) High-magnification view of the dorsal hinge region of a rn-lacZ wing disc stained for β-galactosidase and SoxF transcription (fluorescent in situ hybridization). (G') Confocal cross-section though the dashed line in G. The expression of SoxF abuts the rn-lacZ domain. (H) wg-lacZ (wgZ) disc stained for β-galactosidase and SoxF transcription (fluorescent in situ hybridization). SoxF transcription abuts the wg IR (arrowhead). In addition, SoxF overlaps the wg OR (arrow). (I) Schematic representation of the pattern of expression of SoxF relative to tsh, nub, rn and wg. The expression of SoxF straddles the gap domain delimited by Tsh and Nub. All discs (except in B) are with dorsal up and posterior to the right.

View larger version (68K): [in this window] [in a new window]   Fig. 2. Loss of SoxF function leads to overproliferation of hinge cells without repatterning. (A,B) Wild-type (A) and SoxF mutant (B) late L3 Drosophila wing discs stained for Nub (green), actin (rhodamine-phalloidin, red), and Tsh (blue). The wing pouch is outlined by the dashed line. The solid line in A and B marks the position of their corresponding cross-sections as shown in A',A'' and B', B'', respectively. In a wild-type disc (A), Nub marks the pouch and distal hinge cells and Tsh is specifically expressed in the proximal hinge and the notum. In SoxFKG09145 mutant discs (B), the prospective hinge is larger and shows extra folds both ventrally and dorsally (arrowheads). (A'-B'') The overgrown tissue mostly comprises the gap domain (compare B',B'' with A',A''). (C,D) Proliferation, as monitored by BrdU incorporation (S-phase marker), in wild-type (C) and SoxFKG09145 mutant (D) discs. A hinge-specific increase in BrdU incorporation (green) is seen in the mutant disc (arrowheads) relative to the wild type. To detect total BrdU signal, C and D are maximum projections of z-stacks of confocal sections. The discs were counterstained with rhodamine-phalloidin (actin, red) to visualize disc morphology.

View larger version (153K): [in this window] [in a new window]   Fig. 3. wg is derepressed in the hinge of SoxF mutant discs. (A,B) wg-lacZ expression in wild-type (A) and SoxFKG09145 mutant (B) L3 Drosophila wing discs, counterstained with rhodamine-phalloidin. The wg-lacZ signal is the projection of a series of confocal z-sections to allow visualization of the full pattern. wg expression spreads through the hinge region of SoxF mutant discs and the signal is stronger, especially in the ventral disc region. In A-E, the arrowheads, arrows and `m' mark wg expression in the distal hinge, proximal hinge and wing pouch margin, respectively. In B, the expanded domain of wg is marked by an asterisk. (C) SoxFKG09145 clone, marked by the absence of GFP (red), shows derepression of Wg (blue). (D,E) spd-fg-lacZ expression in L3 wing discs in a wild-type (D) or MS209>dsSoxF (E) background. This latter genotype knocks down SoxF transcription by inducing in the hinge an interference construct. Lines in D and E indicate the position of the cross-sections shown in D' and E'. As in A and B, the lacZ signal is a projection and the discs are counterstained with rhodamine-phalloidin. In MS209>dsSoxF discs (E), the spd-fg-lacZ pattern in the distal hinge broadens relative to that in the wild type (D). (D',E') Cross-sections. The green line marks the extent of spd-fg-lacZ expression in the distal hinge (DH). The distal hinge fold is also indicated as a reference. (F-G'') Flip-out clones expressing SoxF, induced between 48 and 72 hours AEL, are marked positively by GFP (red). These clones cell-autonomously repress wg-lacZ (F), the hinge-specific wg reporter spd-fg-lacZ (G) and Wg (G). Merged (F,G) and single channels (F',G',G'') are shown.

View larger version (101K): [in this window] [in a new window]   Fig. 4. wg drives proliferation in SoxF mutant hinge cells. (A-C) Late L3 wing discs from control (A), wgspd-fg (B) and SoxFKG09145, wgspd-fg (C) mutant Drosophila larvae, stained for actin (white) and β-galactosidase (wg-lacZ, green). The width of the hinge is visualized as the distance between the wg IR and OR, which are marked by red arrowheads. A notal fold (asterisk) is marked as a reference. (D,E) Similar levels of apoptosis, as detected with anti-activated Caspase 3 (Cas3), are observed in the hinge region (arrowheads) of both SoxFKG09145 (D) and SoxFKG09145, wgspd-fg (E) mutant wing discs.

View larger version (69K): [in this window] [in a new window]   Fig. 5. SoxF transcription is controlled by the wg pathway. Fluorescent in situ hybridization detecting SoxF transcripts (red) in Drosophila wing discs containing clones overexpressing Arm* (A), Wg (B), TCFDN (C) and Rn (E), or in which TCFDN has been overexpressed with a dpp-GAL4 driver (D). Merged (left) and individual signals (right) are shown. Clones are marked negatively by the absence of CD2 (A,B,E) or positively by GFP (C). In the panels on the right, dashed lines delineate the clones (A-C,E). Images in B and C are projections of confocal z-stacks. (A) In some Arm*-expressing clones, SoxF expression is ectopically induced in the notum (1) or pleura (2) in a cell-autonomous fashion (boxed regions are magnified in A1 and A2, respectively). (B-B'') In wg-expressing clones (arrowhead), SoxF expression in induced inside and outside of the clone (dashed line). (C-C'') Disc containing TCFDN-expressing clones. (C',C'') High-magnification view of the region boxed in C, showing a TCFDN clone, detected by the punctate GFP signal (C'). In this experiment, the GFP signal is affected by the fixation and in situ hybridization protocol, allowing just the presence of clones to be detected, not their exact boundaries. In the example shown, the SoxF signal loss (C'') is associated with a TCFDN clone (dashed line indicates inferred clone boundaries). (D,D') dpp-driven expression of TCFDN results in the downregulation of SoxF transcription where the dpp domain intersects the hinge (boxed area is magnified in D'). (E-E'') Rn ectopic expression clone represses SoxF cell-autonomously.

View larger version (19K): [in this window] [in a new window]   Fig. 6. Model of SoxF action in the developing wing disc hinge. The pattern of expression of Wg is tightly regulated in the hinge. SoxF acts to negatively regulate the wg pathway at two levels: (1) it blocks wg transcription through the spd-fg enhancer and (2) it antagonizes the Wg pathway parallel or downstream of Armadillo (Arm). SoxF is also required to maintain cell survival. More distally, Rn blocks the expression of SoxF in order to allow wg expression. The Wg signal produced in the IR activates the canonical pathway: Arm, together with TCF, regulates gene transcription. The activation of the pathway elicits at least three responses: (1) the establishment of a wg autoregulatory feedback loop, (2) the proliferation of hinge cells and (3) the activation of SoxF expression in the hinge. Only the expression and function of wg at the IR are shown.

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