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First published online May 5, 2004
doi: 10.1242/10.1242/dev.01104

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Wingless eliminates ommatidia from the edge of the developing eye through activation of apoptosis

Hua V. Lin, Ana Rogulja^* and Ken M. Cadigan

Department of Molecular, Cellular and Developmental Biology, University of Michigan, Ann Arbor, MI 48109-1048, USA

View larger version (125K): [in a new window]   Fig. 1. Wg signaling eliminates R cell clusters from the edge of the eye through apoptosis. All micrographs are of 42 hours APF eyes. (A-D) Wild-type eye stained for Wg protein (blue), TUNEL (green) and Elav (red). Wg staining is coincident with the apoptotic cells at the edge of the eye. (E-H) Close-up of the eye perimeter in (A-D). Most of the Wg protein is adjacent to the apoptotic cells, with lower levels in the dying cells, some of which are still Elav-positive (arrows). The edge of this eye is slightly curved, making some of the TUNEL-positive nuclei appear to be present in interior Elav-positive photoreceptors in the optical stack (arrowheads). (I-L) GMR-Gal4::UAS-p35 eyes stained as in wild type. Apoptosis is not observed (J) but a large accumulation of Wg protein is observed (I) coincident with photoreceptors (L; arrow indicates one example). (M-P) Clone mutant for the Wg receptors fz and fz2. Clonal marker (LacZ) is shown in blue, TUNEL in green and Elav in red. No apoptosis is observed inside the fz, fz2 clone. (Q-T) Clones mutant for the Wg signaling component pygo stained in red for Wg (Q,R) or Elav (S,T). Clonal marker (LacZ) is shown in green. Wg expression is still present in the pygo clone. Extra R cell clusters in a pygo clone are apparent (arrows) while decaying clusters are seen in the adjacent pygo+ tissue (arrowheads).

View larger version (118K): [in a new window]   Fig. 2. Accumulation of Wg in ommatidial clusters precedes PCD and depends on Wg signaling. (AD) Wild-type eyes showing time course of Wg protein expression (red) and TUNEL (green) at 24 hours (A), 28 hours (B), 32 hours (C) and 36 hours APF (D). At 24 hours APF, Wg is expressed in a uniform ring at the eye's edge. Starting at 28 hours and peaking at 32 hours APF, Wg expression is found in clusters (arrows). These clusters are fading at 36 hours APF. TUNEL-positive nuclei in the eye interior at 24 and 28 hours APF correspond to dying inter-ommatidial cells. TUNEL-positive nuclei begin to appear at the edge at 32 hours APF and increase by 36 hours APF in the Wg-positive clusters. (E-H) 32 hours APF eye containing a clone of pygo stained for Wg (red), Elav (blue) and clonal marker LacZ (green). Wg expression overlaps with Elav outside the clone (arrows) but not in cells lacking pygo. Note that some of the Wg clusters contain Elav-clusters in a different focal plane (arrowheads), suggesting that they are already undergoing apoptosis. (I,J) pygo clone at 48 hours APF stained for TUNEL (green) and clonal marker lacZ (red). No TUNEL-positive nuclei are observed in the clone. (K,L) pygo clone in 54 hours APF pupal eye stained for Elav (green) and clonal marker lacZ (red) showing two perimeter ommatidia inside the clone (arrow) that have not undergone PCD.

View larger version (129K): [in a new window]   Fig. 5. Ommatidia destined to die are often incomplete. (A,D) Wild-type eye at 5 hours APF stained for Bar (red) and Elav (green). Bar is a marker for R1 and R6. Several ommatidia at the edge contained only one positive Bar cell (arrows), while smaller Elav-positive clusters contained none (arrowheads in D). (B,E) GMR-Gal4::UAS-p35 eye at 32 hours APF stained for Elav (green), Prospero (Pros; red) and Wg (blue). Pros is a marker for R7 and Wg for ommatidia that will later undergo PCD. About 20% of the Wg-positive ommatidia do not contain an R7 (data not shown). Most of the Wg-positive ommatidia containing an R7 lack some R cells (arrowheads,) but occasionally they appear to have a full complement (arrow). (C,F) GMR-Gal4::UAS-p35 eye at 32 hours APF stained for Cut (red) and Wg (green). Cut is a marker for cone cells. Some Wg-positive ommatidia had four cone cells of normal or near normal size (arrows, compared with more interior ommatidia), but most four cone cell clusters appeared smaller than normal (arrowheads). Often fewer than four cone cells were observed per ommatidia (asterisk). (G) SEM of adult control eye showing the periphery of the ventral eye. (H) SEM of an adult eye containing a large fz, fz2 clone covering the ventral portion of the eye showing three smaller ommatidia never seen in controls (arrows).

View larger version (136K): [in a new window]   Fig. 3. Ectopic Wg expressed at 24 hours APF can induce R cell PCD. GMR-Gal4::UAS-wgts eyes were stained for TUNEL (green) and Elav (red). Animals were reared at 25°C (where wgts is inactive) and shifted to 16.5°C at 24 hours APF (A,B), 30 hours (C) or not shifted (D). After the shift to the lower temperature (activating Wg activity), animals were cultured until the equivalent of 42 hours APF (development occurs 2.7x slower at the lower temperature). A shift down at 24 hours APF results in considerable apoptosis in the interior R cells (A,B) while less TUNEL-positive cells are observed when shifted at 30 hours APF. No ectopic PCD was observed in eyes cultured continuously at 25°C (D).

View larger version (128K): [in a new window]   Fig. 4. Requirement of hid, grim and rpr for perimeter apoptosis. All eyes are 42 hours APF except where noted. (A,B) hidP mutant clone stained for TUNEL (green) and clonal marker LacZ (red). No detectable reduction in apoptosis is observed. (C,D) Df(3L)X25 deficiency clone stained as for (A,B). In one clone there is little reduction in TUNEL (arrow), while a strong reduction is observed in the other (arrowhead). (E-H) Df(3L)H99 clone stained for clonal marker LacZ (blue), TUNEL (green) and Elav (red). Very little TUNEL is observed inside the clone (F) and extra R cell clusters are apparent (arrows in G). (I,J) Df(3L)H99 clone at 30 hours APF stained for Wg (red) and clonal marker lacZ (green). Wg-positive ommatidia are present inside the clone. (K,L) Df(3L)H99 clone at 54 hours APF stained for Elav (red) and clonal marker GFP (green). Perimeter ommatidia are still observed inside the clone at this late time. (M-O) GMR-Gal4::UAS-p35 eyes stained for hid (M), grim (N) or rpr (O) transcripts. Elevated expression of all three genes is observed at the edge of the eye. (P-R) GMR-Gal4::UAS-GPI-fz2 eyes stained for hid (P), grim (Q) or rpr (R). No elevated expression of these genes was observed at the edge of the eye.

View larger version (169K): [in a new window]   Fig. 6. The R cell apoptosis in apc1 mutants is absent in H99 clones. All micrographs are of the interior of eyes at 42 hours APF. (A-C) Wild-type eye stained for Elav (red) and TUNEL (green). No PCD is apparent. (D-F) apc1S76/apc1Q8 mutant eye stained as above. Many of the Elav-positive cells are TUNEL-positive. (G-I) Large Df(3L)H99 mutant clone in an apc1S76/apc1Q8 mutant background stained as above. No cells outside the clone are visible in the field shown. There is a complete block of apoptosis and the Elav pattern is identical to wild type.

View larger version (36K): [in a new window]   Fig. 7. Cartoon of Wg-induced PCD at the eye's perimeter. Non-ommatidial cells at the edge of the eye are depicted as oblongs and ommatidial cells as circles. A red outline indicates Wg expression and purple represents hid, grim and rpr expression. At 24 hours APF, Wg is found only in the edge cells. Between 28-32 hours APF, Wg expression is induced in the perimeter ommatidia, which are often lacking the full complement of cells. Wg signaling is necessary but not sufficient for this induction of ommatidial Wg expression. By 36 hours APF, Wg expression in the ommatidia is fading and the expression of the pro-apoptotic genes hid, grim and rpr are triggering apoptosis, causing the elimination of these ommatidia. See text for further details.

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