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First published online December 1, 2003
doi: 10.1242/10.1242/dev.00884

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Wg and Egfr signalling antagonise the development of the peripodial epithelium in Drosophila wing discs

Luis Alberto Baena-López^*,, José Carlos Pastor-Pareja^*, and Jaime Resino^*

Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Facultad de Ciencias, 28049, Madrid, Spain

View larger version (69K): [in a new window]   Fig. 1. Development of the peripodial side of the wing disc. (A) PE structure in a third instar wing disc. Characteristic low-density nuclear distribution and squamous cell morphology are noticed in the PE. Nuclei are stained with the DNA dye TO-PRO-3 (red) and Arm localization in the zonula adherens (white) reveals cell shape. A white shadow delimits the surrounding cubic cells. The different cell types are described in a transverse section of a disc. (B-D) Ubx expression in the developing PE. In mid second instar (B), major differences in cell shape throughout the disc are not observed. In late second instar (C), squamous cells are seen in a proximal territory of the peripodial side of the disc (arrowheads). At this time, the expression of Ubx is still in poor correlation with squamous morphology. GFP expression in the wing-notum side of second instar discs, driven by ap-GAL4, allows comparison of cell shape between the two sides of the disc in the transversal sections. In third instar discs (D), Ubx is expressed in squamous and some cubic cells. Notice the decreasing gradient of Ubx expression in cubic cells (brackets and inset at higher magnification). (E-G) zfh-2 (green) and iro-C (red) expression domains in developing wing discs. In early mid second instar (E), zfh-2MS209 GAL4 (see Materials and methods) and iro-C are expressed in proximal and distal cubic cells, respectively. Note that in early third instar discs (F) zfh-2 and iro-C domains now overlap in cubic posterior cells (arrowhead). During late larval development (G), iro-C-lacZ expression appears in some distal cubic cells (arrowhead). Discs are oriented anterior towards the left and distal upwards in all panels and figures.

View larger version (113K): [in a new window]   Fig. 3. Ectopic Wg signalling transforms the identity and cell morphology of the PE into wing hinge. (A) wg-expressing clones (green, 48-72 hours AEL) transform both cell autonomously and non-autonomously squamous peripodial cells into columnar cells, as seen both in a surface view and a longitudinal section of the outlined clone. Arm (red) and TO-PRO-3 (blue) staining reveal cell shape and tissue structure (apical membranes of both sides of the disc separated by a dotted line in the section s1). The arrowheads in the section indicate clones in the wing-notum side that do not transform the shape of apposed squamous cells. (B) The transformations extend 7-15 cell diameters away from the clone (green, 48-72 hours AEL). The severity of the transformation decreases the further the affected cell is from the clone (see inset). (C) These clones (48-72 hours AEL) eliminate Ubx expression (red) in peripodial transformed cells (outlined clone and inset) and cubic cells (arrowhead). (D) By contrast, in Arm overexpression clones (24-48 hours AEL) both the transformation in shape and the elimination of Ubx expression are only cell autonomous (inset). wg expressing clones (green) induce expression of hinge markers in the peripodial side of the wing disc, such as ds (E), zfh-2 (F) and nub (G). (E) In these clones ds (red) is induced in cells both inside and outside the clone (see longitudinal section s2). Clone generated 24-48 hours AEL. (F) Clones in squamous (outlined clone) and proximal cubic cells (arrowhead) induce cell autonomous (see transversal section s3) and non-autonomous expression of zfh-2 (red). Clone generated 24-48 hours AEL. (G) Ectopic Wg (48-72 hours AEL), however, induces only autonomous expression of nub (red), which is expressed in the wild-type wing blade and proximal hinge. Notice that the expression of Nub induced is mostly cytoplasmic.

View larger version (111K): [in a new window]   Fig. 2. Wg and Egfr signalling pathways are not required for the development of the PE. (A-C) Clones lacking Wg signalling because of E-Cadhintra5 overexpression (green). These clones accumulate the Wg signalling transcriptional effector Arm (red) in the cytoplasms (A, 24-48 hours AEL). The arrowhead indicates the region of the clone magnified in the insets. (B) E-Cadhintra5 overexpression reduces the proliferation and survival of wing-notum cells (arrowheads), but have no effect in peripodial cells (outlined clone). Wg is detected in red. Clones generated 24-48 hours AEL. (C) These clones also eliminate the expression of zfh-2 (red) in cubic cells (inset at higher magnification of the outlined clone). Clones generated 48-72 hours AEL. (D) Clones lacking Egfr signalling because of overexpression of DN-Raf3.1 (green, 24-48 h AEL) reduce iro-C expression (red) in cubic cells (inset at higher magnification of the region pointed by the arrowhead). The same clone does not affect the proliferation and survival of squamous cells (arrow).

View larger version (125K): [in a new window]   Fig. 4. Ectopic co-expression of wg and vg transforms the PE into wing blade. (A) wg-vg expressing clones (green, 24-48 hours AEL) transform squamous cells into columnar wing blade cells (see longitudinal section s1 through the outlined clone). Vg (blue) is present only in cells of the clone, while nub (red) is expressed both cell autonomously and non-autonomously. (B) wg-vg expressing clones generated 24-48 hours AEL induce the expression of the hinge marker zfh-2 in an exclusively non-autonomous way (see transverse section s2). The arrowheads indicate a clone in the wing-notum side that neither induces zfh-2 expression in apposed peripodial cells nor affects their morphology. Note that the clone in the peripodial side does not induce zfh-2 expression in the wing-notum side either. Arrows indicate untransformed peripodial nuclei. (C) Clones generated 48-72 hours AEL promote autonomous expression of the wing pouch genetic marker dll. In contrast to earlier clones, the induced expression of nub is exclusively non-autonomous (insets at higher magnification).

View larger version (132K): [in a new window]   Fig. 5. Ectopic Egfr signalling transforms the identity and cell morphology of the PE into notum. (A) vn-expressing clones (green) transform the cell morphology of peripodial cells in a incompletely penetrant way (asterisk in a region of the clone not transformed). The transformation affects cells outside the clone (arrowhead). Ubx expression is repressed in some regions of the clone. (B) Expression of the constitutive Egfr activator RasV12 (clones in green) transforms both cell autonomously and non-autonomously squamous peripodial cells into columnar cells, as seen both in a surface view and a longitudinal section (s1). Arm (red) and TO-PRO-3 (blue) staining reveal cell shape and tissue structure (apical membranes of both sides of the disc separated by a broken line). (C) RasV12-expressing clones eliminate Ubx expression (red) in transformed cells inside, but not outside the clone (inset at higher magnification of the outlined clone). An asterisk marks the region of non-autonomous transformation, not fully penetrant around the clone. The arrowhead indicates a clone in the wing-notum side that does not affect apposed squamous cells. (D) iro-C is induced autonomously by RasV12 expression in peripodial cells (longitudinal section s2 of the outlined clone). Asterisk indicates a clone in the wing-notum side of the disc that does not affect apposed squamous cells. (E) RasV12 induces expression of iro-C in distal cubic cells (inset at higher magnification of the outlined clone). The clones in all panels were generated 48-72 hours AEL.

View larger version (34K): [in a new window]   Fig. 6. (A-C) Schematic models of the effects of Wg and Egfr activities in the PE. (A) The squamous morphology and genetic specification of peripodial cells (A) is transformed by ectopic Wg (B) or Egfr (C) signalling. (D-G) Different mechanisms that could account for the absence of Wg and Egfr signalling in the PE (see Discussion). The decay in the concentration of the ligands from their sources in the wing-notum side could lower this concentration down to zero (D) or below a hypothetical activation threshold (E). Alternatively, peripodial cells could be refractory to Wg and Egfr activities because of repression of the signals downstream of the receptor level (F) or decreased diffusive ability of the ligands in peripodial cells (G). These two latter possibilities imply a previous genetic heterogeneity that sets the limits of the peripodial developmental field from early larval stages and implements suppression of Wg and Egfr signalling (H).