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First published online December 1, 2003
doi: 10.1242/10.1242/dev.00904

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Wingless and Notch signaling provide cell survival cues and control cell proliferation during wing development

European Molecular Biology Laboratory, Meyerhofstraße 1, 69117 Heidelberg, Germany

View larger version (42K): [in a new window]   Fig. 1. Wg signaling influences growth during wing development. (A,B) Confocal sections of third instar wing imaginal discs labeled with antibodies to visualize Wg (red), Nubbin (green) and the Myc epitope tag (blue) (A) Wild type. D and V indicate dorsal and ventral compartments. (B) Reduction of Wg signaling in the D compartment of an ap-Gal4 Uas-nkd-myc (II) wing disc. (C-E) Wing discs with multiple marked clones. Clones were marked by the absence of nuclear GFP (C,D) or ß-galactosidase (E). The twin-spots express two copies of the marker and appear brighter. (C) Control clones genotype: hsFLP122/+; FRT42D arm-lacZ/FRT42D Ubi-GFP. (D) Genotype: hsFLP122/+; FRT42D arrow2/FRT42D Ubi-GFP. (E) Genotype: hsFLP122/+; FRT82B pygoS28/FRT82B arm-lacZ. (F-H) Histograms presenting the areas of mutant and twin spot clones (x axis, thousands of pixels). Clones in the wing pouch and notum are shown separately. Individual pairs of twin spots and mutant clones are in the same order (shown according to the increasing sizes of the twin). (F) Wild-type clone average area was 1736 versus 1706 pixels for the twin spots (n=25) in with wing pouch. In the notum, average areas were 2046 and 2002 pixels (n=11). (G) arrow twin versus clone, 2406 versus 419 pixels (n=29) in the wing pouch, and 1204 versus 737 pixels (n=14) in the notum. (H) pygoS28 twin versus clone areas were 2036 versus 950 pixels (n=24) in the wing pouch, and 1796 versus 1120 pixels (n=14) in the notum.

View larger version (88K): [in a new window]   Fig. 2. Apoptosis in cells with reduced Wg signaling. (A,B) wing disc with arrow2 mutant clones. (A) Disc labeled to visualize apoptotic cells with an antibody to activated caspase-3 (red). Clones were marked by the absence of GFP (green). Nuclei were labeled with DAPI (blue). Apical (left) and basal (right) optical sections are shown for the same disc. (B) Projection of four sections from the disc in A taken at 5 µm intervals, showing anti-capsase labeling. Clones are outlined. (C) pygoS28 clones marked by the absence of ß-Gal (green). (D) Projection of four sections from the disc in C.

View larger version (159K): [in a new window]   Fig. 3. JNK pathway activation in arrow2 mutant clones. (A) Apical and basal optical sections of a wing disc with arrow2 mutant clones. Clones were marked by the absence of GFP (green). Apoptotic cells were labeled using an antibody to activated caspase-3 (blue). puc-LacZ expression was visualized with anti-ß-Gal (red). (B) Magnification of the central region of the wing pouch of the disc in A. Lower panels show puc-lacZ and cleaved caspase-3. Scale bar: 25 µm.

View larger version (114K): [in a new window]   Fig. 5. Effects of reduced cell competition. (A,B) Wild type and pygoS28 Minute+/+ in a Minute+/- heterozygous background. Clones were labeled by the absence of ß-gal. (C) Histogram comparing sizes of the control and pygo mutant clones. Average areas were 2096 pixels (n=140) and 1236 pixels (n=55), respectively. (D,E) Wild-type and arrow2 Minute+/+ in a Minute+/- heterozygous background. Clones were labeled by the absence of Myc. The inset in E shows pyknotic nuclei in the basal region of the arrow2 clone labeled with DAPI. (F,G) Wild-type and pygoS28 Minute+/+ in a Minute+/- heterozygous background. Activated caspase-3 is shown in red in a projection of six single sections taken at 3.5 µm intervals. The clones are shown as a single channel of an apical section of the same image in green. Note the similar levels of cleaved caspase-3 in wild-type and pygoS28 mutant clones. Apoptotic cells were mainly found in the Minute+/- heterozygous cells adjacent to Minute+/+ wild-type cells, as a result of cell competition. (H-K) Wing disc with both arrow2 and pygoS28 mutant clones. Clones were labeled by the absence of GFP and ß-gal. Dll is shown in blue. Note the lower levels of Dll present in arrow2 clones (green) compared with pygoS28 clones (red).

View larger version (108K): [in a new window]   Fig. 4. Clonal growth in the absence of cell death. Wing discs with clones of cells expressing p35 or Puckered labeled by co-expression of CD8-GFP (green); antibody to activated caspase-3 (red). Nuclei were labeled with DAPI (blue). (A) Wild-type clones expressing p35. (C-E) arrow2 mutant clones expressing p35. (E) Magnification of the disc in D. Note the absence of pyknotic nuclei and cytoplasmic staining of caspase-3 when p35 is expressed. Scale bar: 25 µm. (F) Wild-type clones expressing Puckered. (H-J) arrow2 mutant clones expressing Puckered. (J) Magnification of the disc in (I). (B,G) Histograms comparing sizes of control and arrow mutant clones co-expressing p35 (B) or Puc (G).

View larger version (76K): [in a new window]   Fig. 7. Notch and Wg effects on cell proliferation and cell cycle phasing. (A-D) Wing imaginal discs labeled by incorporation of BrdU (red) to mark cells that have gone through S phase (projection of four optical sections taken at 5 µm intervals). BrdU channel is shown separately below. Clones were marked by the expression of GFP (green). (A) Wild-type clones expressing CD8-GFP. (B) Clones expressing NotchIntra and CD8-GFP. (C) Clones expressing NotchIntra and CD8-GFP, and mutant for wg. (D) axin mutant clones marked by expression of GFP. (E) Flow cytometric analysis of cells of the genotypes shown in A-D. Co-expression of GFP was used to sort mutant cells. (F) Flow cytometric analysis of cells expressing p35 and mutant for arrow.

View larger version (120K): [in a new window]   Fig. 8. Notch and Wg effects on cyclin E expression. (A) Cyclin E expression (red) in a wild-type disc containing clones of cells expressing GFP (green). (B) Reduced cyclin E expression in clones expressing activated Notch and GFP. (C) Reduced cyclin E expression in clones mutant for axin and expressing GFP. Lower panels show the GFP and cyclin E channels separately.

View larger version (88K): [in a new window]   Fig. 9. Effects of Notch on growth, dependent and independent of Wg. (A-C) Clones expressing UAS-Notchintra labeled with CD8-GFP (green), produced using the Gal4/Gal80 system. Wg protein (red); Vestigial (Vg) protein (blue). (A) Otherwise wild-type clones. (B,C) Clones also mutant for wgcx4. Clones expressing Notchintra were not able to induce overgrowth in the wing pouch in the absence of Wg expression, but did induce overgrowth in the wing hinge and pleura. (C') Detail of panel C. (D) Histogram comparing the percentage of Notchintra-expressing clones able to induce tissue overgrowth in different areas of the wing pouch and in the wing hinge in the presence or the absence of wg. Their ability to induce tissue growth was dependent on their position. The dark gray bar indicates clones that induced overgrowth. The number of clones examined is shown in below. The diagram to the right indicates the different areas used for quantification.

View larger version (84K): [in a new window]   Fig. 6. Notch and Wg induce cell proliferation. (A-C) Wing imaginal discs labeled by incorporation of BrdU to mark cells that have gone through S phase (red). Nuclei were labeled with DAPI (blue). Cells expressing dpp-Gal4 and UAS-GFP were labeled with anti-GFP (green). (A) dpp-Gal4 UAS-GFP disc, (B) dpp-Gal4, UAS-GFP and UAS-Notchintra, and (C) dpp-Gal4 UAS-GFP and UAS-Wg. Note the non-autonomous induction of BrdU incorporation near the GFP-expressing cells in B and C.