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First published online November 7, 2006
doi: 10.1242/10.1242/dev.02686

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Lobe and Serrate are required for cell survival during early eye development in Drosophila

¹ Department of Molecular and Cellular Biology, One Baylor Plaza, Houston, TX 77030, USA.
² Program in Developmental Biology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.
³ Department of Ophthalmology, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030, USA.

View larger version (69K): [in a new window]   Fig. 1. Loss of L function causes selective induction of cell death in the ventral eye. (A,A') Wild-type eye imaginal disc showing a few randomly distributed TUNEL-positive nuclei of dying cells. (B,B') L-mutant eye disc (L2/+), showing TUNEL-positive ventral eye cells nuclei (arrow). (B,B') Semicircular dashed line marks the lost boundary of the ventral eye field. (C,C') LOF clones of L show TUNEL-positive cells nuclei in the ventral eye (arrow). The cells lacking L gene function in the dorsal half of the eye are not affected (arrowheads). Straight dashed lines in A-C indicate the approximate midline - the border between the dorsal (D) and ventral (V) eye.

View larger version (103K): [in a new window]   Fig. 2. Cell death in L/Ser-mutant cells is p53-independent and caspase-dependent. (A-B') Overexpression of p53 (A) in the wild-type eye using ey-GAL4 (ey>p53) results in a small eye and, in the L2/+-mutant eye (L2/+; ey>p53; B,B'), does not affect the loss-of-ventral-eye phenotype. Dashed line marks the lost boundary of the eye field. (C-D') Blocking caspase-dependent cell death by overexpression of baculovirus P35 in the wild-type eye (C) does not affect eye size whereas, in the L2/+-mutant background (L2/+; ey>p53), this can significantly rescue the loss-of-ventral-eye phenotype in the eye imaginal disc (D) and adult eye (D'). (E-H') Overexpression of diap1 (ey>diap1; E) or a reduction in the levels of the Hid-Reaper-Grim complex by using deficiency H99 (G) in wild-type eye does not affect eye size, whereas, in L2/+-mutant backgrounds (F,F',H,H'), these can rescue the loss-of-ventral-eye phenotype. (I) Overexpression of dominant-negative Ser (ey>SerDN) results in loss of ventral eye (Singh and Choi, 2003). Dashed line marks the lost boundary of the eye field. (J,J') Blocking caspase-dependent cell death can significantly rescue the loss-of-ventral-eye phenotype of dominant-negative Ser overexpression (ey>SerDN+P35), as seen in eye disc (J) and adult eye (J'). Markers for immunostaining are shown in color labels.

View larger version (119K): [in a new window]   Fig. 3. Canonical Wg signaling pathway affects the L-mutant phenotype. (A-D') Increasing levels of canonical Wg signaling in the eye by overexpressing Wg (A) or Arm (C) results in small eyes. However, in the L2/+-mutant background, overexpression of Wg (B,B') or Arm (D,D') results in the enhancement of loss-of-ventral-eye to a no-eye phenotype. (E-H') Reducing Wg signaling by overexpressing Sgg (E) or dTCFDN (G) in wild-type eye does not affect eye size. However, in the L2/+ background, overexpression of Sgg (F,F') or dTCFDN (H,H') strongly suppresses the loss-of-ventral-eye phenotype to near that of wild-type eye. Dashed line marks the lost boundary of the eye field. AN, antenna.

View larger version (109K): [in a new window]   Fig. 4. Wg acts antagonistically to L. Loss of L (L2/+) results in preferential elimination of the ventral eye cells, as seen in eye disc (A), adult eye (B) and adult eye sections (C,C'). The loss-of-ventral-eye phenotype of L2/+ mutants is restored in the wg-mutant background (L2, wg1/CyO), as seen in the eye disc (D) and adult eye (E). Sections of L2, wg1/+ fly eye show restoration of the ventral eye-specific ommatidia (F,F'). In C' and F', circles represent ommatidia with uncertain polarity, and blue and red arrows indicate dorsal and ventral polarity, respectively. In third instar eye disc, wg-lacZ is strongly expressed on both dorsal and ventral polar margins (Royet and Finkelstein, 1997). (G) In the L2/+-mutant background, wg-lacZ is ectopically induced on the ventral posterior margin of the eye. (H-I') LOF clones of Lrev, marked by loss of the GFP reporter, in the ventral eye show ectopic induction of Wg (arrow). The size of the ventral eye is smaller, suggesting that some of the ventral eye cells are lost. There is no effect on Wg in LOF clones of L in the dorsal eye (marked by dashed line, arrowhead).

View larger version (90K): [in a new window]   Fig. 5. Cell death in the L-mutant eye is due to ectopic induction of Wg. (A-A''') The LOF clones of L on the ventral eye margin (A, arrow), posterior to MF and marked by the loss of GFP reporter (A', arrow), show ectopic induction of Drice (A'', arrow), coincident with the ectopic induction of Wg (A''', arrow). (B,C) LOF clones of L, generated by the MARCM approach, are positively marked by the GFP reporter and accompanied by the overexpression of SggS9A (B) or dTCFDN (C) to abolish Wg signaling in the eye. Notice that LOF clones of L in the ventral eye can no longer eliminate eye pattern (arrowheads).

View larger version (87K): [in a new window]   Fig. 6. L is involved in the repression of wg during second larval instar. (A-C) Abolishing wg function in different time windows using a temperature-sensitive wgIL114 allele (details in Materials and methods) shows that second instar of larval development (L2) is crucial for L-Wg interaction. (B) When wg function was abolished during first or third instar, there was no significant rescue of the L-mutant phenotype. (C) When wg function was abolished during second instar, loss-of-ventral-eye phenotype of L2/+ mutants was significantly rescued to near that of wild-type eye. (D) In second instar wild-type larval eye disc, wg-lacZ is expressed strongly on the dorsal margin. (E) In the L2/+-mutant background, a strong induction of wg-lacZ reporter expression is seen all along the ventral polar margin. Dashed lines in D,E mark the boundary between the dorsal- and the ventral-half of the eye. (D,E) Inset shows wg-lacZ expression (red) in a separate channel in second instar eye imaginal discs. (F) In the second instar eye disc, LOF clones of L cause ectopic induction of Wg and Drice in the ventral eye (arrowhead). Separate channels are shown in inset.

View larger version (98K): [in a new window]   Fig. 7. Loss of L gene function results in activation of the JNK signaling pathway. (A) In L2/+ eye disc, puc-lacZ, a reporter for JNK signaling pathway activation, is ectopically induced on the ventral eye margin. (B,B') In LOF clones of L, ectopic induction of puc-lacZ was observed in only the ventral eye margin (arrows). In the dorsal eye clones, puc-lacZ was not induced (arrowheads). (C-D') Blocking JNK signaling by overexpression of puc (Martin-Blanco et al., 1998) in the eye does not affect eye size (C), whereas, in L2/+-mutant eyes, it rescues the ventral eye loss in the eye imaginal disc (D) and adult eye (D'). (E) Overexpression of a dominant-negative form of Bsk (BskDN) alone in the eye served as control. (F,G) Reducing levels of JNK signaling by overexpressing BskDN (F) or by using a heterozygous background of a null mutant of hep (hep/+; L2/+; G) rescues the L-mutant eye phenotype. (H-I') Increasing levels of JNK signaling by overexpressing the activated form of Bsk (BskAct) in wild-type eye (H) results in small eye but, in L2/+-mutant eye (I,I'), it causes severe enhancement of the loss-of-ventral-eye phenotype. (J-K') Overexpression of the activated form of Drosophila Jun in the wild-type (J) does not affect the eye. However, overexpression of Drosophila Jun strongly enhances the L2/+-mutant phenotype (K,K') in the eye. Dashed lines mark the lost boundary of the eye field. AN, Antenna.

View larger version (78K): [in a new window]   Fig. 8. L-mutant phenotype is dependent on activation of both JNK signaling and caspase-dependent cell death. Blocking caspase-dependent cell death and the JNK signaling pathway in the wild-type eye (ey>P35+puc) (A) have no significant effect on eye development, whereas, in the L2/+ mutant (L2/+; ey>P35+puc) it results in the strong rescue of the loss-of-ventral-eye phenotype in the eye disc (B) and adult eye (B'). (C) Graph showing more than 1.5-fold increases in the frequency of rescue of the L-mutant phenotype by blocking both caspase-dependent cell death and JNK signaling as compared with blocking either one of them alone.

View larger version (17K): [in a new window]   Fig. 9. Model for L and Ser function in cell survival during early eye development. L and Ser are required for cell survival during early eye development by preventing the ectopic induction of Wg- and JNK-signaling pathways in the ventral eye or in the entire eye disc prior to the establishment of the DV pattern. L and Ser can inhibit the JNK signaling pathway indirectly by repressing Wg signaling. It is also possible that L and Ser directly block JNK signaling. JNK-signaling activation can induce both caspase-dependent and -independent cell death. Thus, L and Ser promote cell survival by blocking both JNK-signaling-mediated and caspase-dependent cell death.

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