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A pathway of signals regulating effector and initiator caspases in the developing Drosophila eye

Sun-Yun Yu1, Soon Ji Yoo2, Lihui Yang1, Cynthia Zapata1, Anu Srinivasan3, Bruce A. Hay2 and Nicholas E. Baker1,*

1 Department of Molecular Genetics, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
2 Division of Biology, California Institute of Technology, 1200 East California Boulevard, Pasadena, CA 91125, USA
3 IDUN Pharmaceuticals, 11085 N. Torrey Pines Road, Suite 300, La Jolla, CA 92037, USA



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  		Fig. 1. Embryos labelled with CM1 antiserum. Anterior towards the left in these and subsequent figures. (A) Wild type, stage 12. (B) Wild type, stage 15. (C) In western blots, CM1 recognised cleaved Drice but not cleaved Dronc or DCP1. (D) Alignment of the putative p20/p10 linker regions from Drosophila caspases with the human caspase 3 peptide and with caspase 1 from S. frugiperda, for which cleavage has been demonstrated C-terminal to either aspartate residue (Ahmad et al., 1997Go). Residues identical to the 13-mer CM1 peptide shaded (gaps has been introduced to permit alignment of active sites and p20 C termini). Long pro-regions suggest Decay, Damm, Dredd, Dream and Dronc are likely to be regulated initiator caspases.

 


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  		Fig. 2. (A,B) Wild-type retinas labelled with CM1 antiserum. (A) 30 hours APF. (B) 43 hours APF. (C,D) GMRp35 retinas labelled with CM1 antiserum. (C) 30 hours APF. (D) 43 hours APF. (E,F) Enlargement of wild-type retina, 30 hours APF. (E) Apical. (F) Basal. Most labelled cells are apoptotic and basal. A minority also extend apically and are more normal morphologically (arrows). (G,H) Enlargement of GMRp35 retina, 30 hours APF. (G) Apical. (H) Basal. All the labelled cells are morphologically typical of the pigment cell lattice and extend from apical to basal. Note that activated caspases are excluded from the nuclei. (I) GMRp35 retina (43 hours APF) labelled with CM1 antiserum (green) and for Notch protein (magenta). (J) Eye from Elp/Elp adult. (K) Eye from Elp/Elp; GMRp35 adult. Facet number is comparable with J, but the pigmented, unfaceted region is much larger. (L) Enlargement of Elp/Elp eye disc. Weakly labelled cells are first seen around column 5. Labelling intensifies and cells progressively round up and fragment until most labelled cells have been eliminated posterior to column 15. (M) Enlargement of Elp/Elp; GMRp35 eye disc, apical plane. Weakly labelled cells are first seen around column 5. Labelling intensifies around the cell boundaries and highlights the normal cellular morphology. Labelled cells are maintained in the disc, not lost from the epithelium. Note that ommatidia (arrow) are protected. (N-Q) Eye imaginal discs labelled with CM1 antiserum. (N) Elp/Elp. Most cells in columns 7-15 are apoptotic. (O) Elp/Elp; GMRp35. Most undifferentiated cells posterior to the furrow are labelled. Occasional ommatidia and surrounding cells are unlabelled. In contrast to N, labelled cells are not lost from the back of the disc. (P) Oregon R (w). Scattered cell deaths are seen posterior to column 7. (Q) GMRp35. Cells protected from cell death label with CM1 but do not have apoptotic morphology.

 


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  		Fig. 3. Cell death in wild-type eye discs. GMRp35/+ eye discs labelled with the CM1 antiserum. Regions of moderate labelling vary in location between individuals. (A,B) Oregon R background. (C,D) Canton S background.

 


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  		Fig. 4. Pupal retinas labelled with CM1 antiserum 30 hours APF. (A) Wild type. (B) GMRdronc/+. (C) GMRdroncDN/+. (D) GMRp35/+. (E) GMRp35/GMRdronc. (F) GMRp35/GMRdroncDN. (G) GMRDIAP1. (H) GMRDIAP1/GMRp35.

 


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  		Fig. 5. Scanning electron micrographs of adult eye surfaces. (A) GMRp35. Mild eye roughening is manifested by subtle variations in facet size and shape and orientation of interommatidial bristles. (B) GMRDIAP1/+. Eye morphology was normal despite suppression of cell death (Hay et al., 1995Go). (C) GMRDIAP1/GMRp35. Eye morphology was rescued compared with GMRp35. If eye roughness was caused by survival of supernumerary cells, eye roughness would be expected to be enhanced or unaffected by co-expression of DIAP1 and p35. If p35 had effects independent of cell survival, eye roughness would be expected to be unaffected by co-expression of DIAP1 and p35. If Dronc activity perturbed eye development independently of cell survival, eye roughness would be expected to be reduced by co-expression of DIAP1 and p35 because DIAP1 inhibits Dronc (Hawkins et al., 2000Go; Meier et al., 2000bGo). (D) GMRDronc-DN/GMRp35. Eye morphology was rescued when compared with GMRp35, as predicted if Dronc activation were responsible for the eye roughening that occurred when p35 blocked cell death. (E) GMRDronc/GMRp35. Mild eye roughening was similar to GMRp35. (F) GMRDronc/+. Eye morphology was normal. (G) GMRDronc-DN/+. Eye morphology was normal. (H) GMRDronc/GMRDIAP1. Eye morphology was normal. (I) GMRDronc-DN/GMRDIAP1. Eye morphology was normal.

 


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  		Fig. 6. Pupal retinas labelled with CM1 antiserum 30 hours APF (25°C). (A) Wild type. (B) hid /hid. (C) egfrts1a /egfrf24, 31.5°C for 4 hours. (D) Nfag. (E) Nl1-Nts1, 31.5°C for 6 hours. (F) Nl1-Nts1; egfrtsla /egfrf24, 31.5°C for 4 hours. (G) Nfag;egfrtsla /egfrf24, 31.5°C for 4 hours.

 


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  		Fig. 7. Eye imaginal discs labelled with CM1 antiserum. (A) hid/hid. (B) hid/+. (C) GMRDIAP1 /GMRDIAP1. (D) GMRDIAP1/GMRp35. (E) Nl1-Nts1, 31.5°C for 4 hours. (F-H) egfrf24 clones in hid/hid larva. (F) ArmlacZ. Arrows indicate the small clones that result when Minutes are not used. (G) CM1 labelling. (H) F,G combined, showing complete absence of apoptosis from clones (arrows). (I-L) egfrf24 clones in GMRDIAP1 larva. (I) ArmlacZ. (J) CM1 labelling. (K) ELAV labelling of differentiating photoreceptors. (L) Combination of I-K showing apoptosis of egfr mutant cells. (M-P) egfrf24 clones in GMRp35 larva. (M) ArmlacZ. (N) CM1 labelling. (O) ELAV labelling of differentiating photoreceptors. (P)M-O combined. Contrast morphology of CM1-labelled cells with the apoptotic cells in J. Note reduced CM1 labelling of differentiating egfrf24 photoreceptors (arrows in N,P).

 


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  		Fig. 8. Cell death pathways in the Drosophila retina. Levels of activated effector caspases depend on two opposing pathways. Inhibitor of apoptosis proteins (DIAPs) block caspase activation. Initiator caspases promote caspase activation. EGFR-dependent survival signalling acts through DIAPs by inhibiting the HID, a DIAP inhibitor. A pro-apoptotic signal mediated by N also acts through this pathway, antagonising EGFR-dependent survival signalling. N has this role only in pupal retina, not in eye imaginal discs, and is antagonised by primary pigment cells and/or cone cells. Thus, caspase activity is blocked by multiple extracellular signals. Dronc and p35-sensitive initiator caspases act redundantly for effector caspase activation in most eye cells. No extracellular signals have yet been found that promote caspase activation directly and it is possible that no such regulation exists, although our results raise the possibility that in eye disc cells Hid might activate caspases independently of DIAPs. In addition, our results indicate that Dronc has effects in dying cells independent of effector caspases. The data do not rule out more elaborate models in which EGFR is also a receptor for primary pigment cells and/or cone cell survival signals upstream of N, as well as acting downstream of N. However, such models require that EGFR function upstream of N depends on primary pigment cells and/or cone cells whereas EGFR function downstream of N does not.

 

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© The Company of Biologists Ltd 2002