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First published online 20 August 2003
doi: 10.1242/dev.00719

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Egfr/Ras pathway mediates interactions between peripodial and disc proper cells in Drosophila wing discs

Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad, India 500 007

View larger version (70K): [in a new window]   Fig. 1. Counting peripodial membrane cells. Wing discs of different genotypes were stained for Armadillo and large peripodial cells were counted on a laser-scanning confocal microscope at 60x magnification. (A) A single optical section of only peripodial membrane. Such optical sections were used to obtain a relative count of peripodial cells in different genetic backgrounds. (B) A single optical section of disc proper. (C,D) vg-GAL4 (C) and 426-GAL4 (D) expression patterns as seen with UAS-lacZ. (E,F) vg-GAL4/UAS-Ubx (E) and 426-GAL4/UAS-Vg (F) wing discs reconstructed from several optical scans showing large peripodial and small disc proper cells.

View larger version (109K): [in a new window]   Fig. 2. Genes/enhancer-trap lines expressed in the wing disc peripodial membrane. (A-D) lio-lacZ (A), odd-GAL4/UAS-nuclear lacZ (B), coro-GAL4/UAS-nuclear lacZ (C) and en-GAL4/UAS-nuclear lacZ (D) wing discs stained with anti-ß-galactosidase antibodies. (E) Wild-type wing disc stained with anti-Ubx antibodies. coro-GAL4 and Ubx are not expressed in the DP, whereas only Ubx is expressed in all PM cells. E also shows spatial domains of the DP. P, pouch; H, hinge, LN, lateral notum; MN, medial notum. (F-J) Ubx-GAL4 expression patterns during different stages of development. The expression of Ubx-GAL4 was detected by either anti-ß-gal antibody staining (F,G,I,J) or X-gal staining (H). (F) In the wing disc, Ubx-GAL4 is expressed (green) only in the peripodial membrane. The disc proper is stained with anti-Wg antibody (red). (G) Stage 15 embryo showing lacZ expression in T2, T3 and in all abdominal segments. (H,I) Mid- to late second instar wing discs, showing PM-specific expression of Ubx-GAL4. In I, the disc proper is stained with anti-Wg antibody (red), which is expressed in the anterior-ventral quadrant of second instar discs. (J) Ubx-GAL4 expression pattern in the haltere disc. It is expressed in the disc proper as well as in the peripodial membrane. This GAL4 driver is predominantly expressed in the entire anterior compartment of the disc.

View larger version (85K): [in a new window]   Fig. 3. Peripodial and disc proper cells are clonally related. Actin5C>lacZ clones were induced by Ubx-GAL4/UAS FLP. All discs were stained with anti-ß-galactosidase antibodies. (A) A wing disc showing clones in both peripodial membrane and disc proper. After confocal imaging, the optical sections were reconstructed using 3D imaging software, which would distinctly show both large peripodial and small disc proper cells. (B-D) Optical sections of a part of the same wing disc showing PM cells (B) and DP cells (C) at higher magnification. The optical section in C focuses on only a few DP cells in the field. The two optical sections were differently colour-coded and merged together to show spatial relationship of PM (green) and DP (red) clones (D). (E-H) Peripodial-only clones. Size of peripodial-only clones varied from just two cells (E) to >128 cells (H). In F-H, there appear to be more than one clone, as medial edge cells are also stained. We did not observe any disc with clone/s only in the disc proper. (I-K) Haltere discs showing both PM and DP clones. In some discs, the relative size of DP clones is much larger than those in wing discs (I-J). For example, in I, DP clone/s cover the entire anterior compartment.

View larger version (114K): [in a new window]   Fig. 4. Ras is required for the viability of PM cells. (A-C) hsFLP; P[FRT] Ras1 Ubi-GFP /P[FRT]82B f+ wing disc with Ras1/Ras1 mitotic clones in the PM. Ras1/Ras1 cells are marked with GFP (A). The disc is also stained for DAPI (B). C is the merge of A and B. Note that the clones are degenerating (note speckled pattern of GFP expression) and are occupied by surrounding normal cells. The regions without GFP expression represent Ras+/Ras+ cells (twin of Ras1/Ras1 clones), which survive normally. (D) ap-GAL4/UAS-nuclear lacZ wing disc stained for ß-galactosidase and Wg expression. ap-GAL4 is expressed in the entire dorsal compartment. (E) ap-GAL4/UAS-Dras1N17 wing disc stained for Wg expression. Note severe reduction in the notum size. (F) ap-GAL4/UASDras1N17; UAS-P35. DN-Ras-induced reduction in notum is partially rescued by P35.

View larger version (110K): [in a new window]   Fig. 5. Reduction in Egfr activity in the PM is sufficient to cause pattern duplications in the DP. (A) Wild-type wing blade. (B-H) Adult wing blades of Ubx-GAL4/UAS-DN-DER (expressed from two copies of the transgene; B), Ubx-GAL4/UAS-Dras1N17 (C-E), Ubx-GAL4/UAS-DN-Raf (F), Ubx-GAL4/UAS-Aos (G) and Ubx-GAL4/UAS-rasN17 (dominantnegative form of mammalian Ras; H) flies. Note pattern duplications (notum-to-wing transformation) in the wing blades and anterior-specific margin bristles in the transformed region. Associated reduction in notum tissue is shown in E. In all wing blades, anterior is towards the top.

View larger version (109K): [in a new window]   Fig. 6. Pattern duplications observed in flies over-expressing DNDER, DN-RAS, DN-Raf and Aos in the PM reflect notum/hinge-to-wing transformations in wing discs. (A-C) Ubx-GAL4/UAS Dras1N17 (A), Ubx-GAL4/UAS-DN-Raf (B) and Ubx-GAL4/UASAos (C) wing discs stained for Wg. Note mesonotum-to-wing blade transformation as indicated by the over-growth and extension of the DV boundary to the mesonotum region (arrowheads). Larvae with such high degree of notum-to-wing transformation are invariably early pupal lethal. (D,E) Ubx-GAL4/UAS-Dras1N17 wing discs double stained with Ci and Wg (D), and En and Wg (E) to show pattern duplications (arrowheads). (F,G) Ubx-GAL4/UAS-Dras1N17 (F) and Ubx-GAL4/UAS-Aos (G) wing discs stained for Ubx. Note that Ubx expression in the PM is not altered in these discs. The figure also suggests that the identity of PM cells is not changed. (H,I) Ubx-GAL4/UAS-Dras1N17 (H) and Ubx-GAL4/UAS-Aos (I) wing discs stained for Arm. Only transformed part of discs are shown at higher magnification. Note that PM cells in these experiments have retained their normal morphology (arrowheads).

View larger version (79K): [in a new window]   Fig. 7. Notum-to-wing transformation observed in Ubx-GAL4/UAS-Dras1N17 or Ubx-GAL4/UAS-Aos was due to their expression in the PM and not in the DP. All wing discs shown in this figure stained for Wg expression. Top panel shows the expression patterns of different GAL4 drivers (visualized with UAS-nuclear lacZ) used in this study. Middle panel shows the effect of overexpression of DN-Ras using those GAL4 drivers and the lower panel shows the effect of overexpression of DN-Aos. 426-GAL4 is expressed only in the presumptive hinge (A), whereas pnr-GAL4 in the presumptive lateral notum (D). Overexpression of DN-Ras (B,E) and Aos (C,F) using these two GAL4 drivers does not affect wing development, reflecting late (early to mid-third instar larval stages) activation of 426- and pnr-GAL4 expression. ap-GAL4 is expressed in the dorsal pouch and in the entire notum (G). Overexpression of DN-Ras (H) or Aos (I) using this GAL4 driver causes severe reduction in notum tissue; however, no transformation is observed. (J-M) tsh-(J) and AgiR-GAL4 (M) drivers are expressed in both DP and PM cells. In the DP, tsh-GAL4 is expressed in the hinge and in the presumptive lateral notum (J), whereas AgiR-GAL4 is expressed in the posterior mesonotum (asterisk in M). Overexpression of DN-Ras (K) and Aos (L) using tsh-GAL4 caused severe loss of notum tissue, and no pattern duplication is observed. DN-Ras overexpression using AgiR-GAL4 resulted in loss of notum tissue (N), whereas no phenotype was observed with Aos (O). (P-R) Ubx-GAL4 expression pattern (P) and the effect of overexpression of DN-Ras (Q) and Aos (R) using this GAL4 driver is shown again (for details, see Fig. 6) for comparison.

View larger version (139K): [in a new window]   Fig. 8. Dynamic expression pattern of Aos in the wing imaginal disc. (A-B) Third instar aos-lacZ wing imaginal disc stained for ß-galactosidase expression (green) and Ubx (red; shown only in B). Aos is expressed in the presumptive veins and in the notum. Note that aos-lacZ and Ubx are not colocalized, suggesting that at this stage of development Aos is expressed only DP cells. Large cells in that notum that express Aos are probably adepithelial cells. (C) Third instar wild type wing disc stained for Ct expression. In the notum Ct is expressed in the myoblasts. (D and D') Part of the notum of aos-lacZ wing imaginal disc stained for ß-galactosidase expression (green) and Ct (red; shown only in D'). Note co-localization of Aos and Ct in the myoblasts. (E,F) Early second instar aos-lacZ wing imaginal discs stained for ß-galactosidase expression (green). Aos is expressed in large PM cells overlaying the notum as well as the pouch. Disc in E is also stained for Ct (red), which marks myoblasts. At this stage during development, myoblasts are localized to the anterior compartment, whereas Aos-expressing cells are in the posterior compartment. (G) Late second instar aos-lacZ wing imaginal disc. (H) Late second instar wild-type wing imaginal disc stained for Ubx expression. Note that Aos and Ubx share a similar expression pattern. (I-K) Late second instar aos-lacZ wing imaginal disc stained for both ß-galactosidase (green; I) and Ubx (red; J) expression. K is the merge of I and K. Note colocalization of Aos and Ubx in PM cells. (L) Early third instar aos-lacZ disc. Aos expression in the pouch (asterisk) begins at this stage during development.

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