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Modes of programmed cell death during Ceratitis capitata oogenesis
Tissue & Cell 35 (2003) 113–119
Modes of programmed cell death during Ceratitis capitata oogenesis Ioannis P. Nezis a , Vassilis Modes a , Vicky Mpakou a , Dimitrios J. Stravopodis a , Issidora S. Papassideri a , Ioanna Mammali b , Lukas H. Margaritis a,∗ a
Faculty of Biology, Department of Cell Biology and Biophysics, University of Athens, Panepistimiopolis 15784, Athens, Greece b Department of Neurology, Aiginition Hospital, University of Athens, Panepistimiopolis 15784, Athens, Greece Received 7 October 2002; received in revised form 27 December 2002; accepted 27 December 2002
Abstract In the present study, we demonstrate the existence of two distinct apoptotic patterns in nurse cells during Ceratitis capitata oogenesis. One is developmentally regulated and normally occurs during stages 12 and 13, and the other is stage specific and is sporadically observed during stages 7 and 8. The pre-apoptotic manifestation of the first pattern begins at stage 11 and is characterized by the formation of actin bundles. Subsequently, at stages 12 and 13, the nurse cell nuclei exhibit condensed chromatin and contain fragmented DNA, as revealed by TUNEL assay. The apoptotic nurse cell remnants are phagocytosed by the neighboring follicle cells at the end of oogenesis during stages 13 and 14. In the second apoptotic pattern, which occurs sporadically during stages 7 and 8, the nurse cells degenerate and are phagocytosed by the follicular epithelium that contains apoptotic cell bodies. The data presented herein, compared to previous reported results in Drosophila melanogaster and Dacus oleae (Nezis et al., 2000, 2001), strongly suggest that nurse cell apoptosis is a developmentally regulated and phylogenetically conserved mechanism in higher Dipteran. They also suggest that, the sporadic apoptotic pattern consists of a possible protective mechanism throughout oogenesis when damaged or abnormal egg chambers, are eliminated before they reach maturity. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Actin cytoskeleton; Apoptosis; Diptera; Oogenesis; Phagocytosis
1. Introduction Oogenesis is a significant biological process that leads to the formation of a highly complex cell, the oocyte. One of its major characteristics is the remarkable gradual increase in the volume of the oocyte due to accumulation of nutrients, mRNAs, proteins and organelles required during early embryonic development. In insects, the acquisition of the above nutritious material can be achieved by several different ways (Gutzeit, 1986). In some insects, including Drosophila melanogaster, the oocyte development is supported by a group of cells, termed nurse cells, which are connected to the oocyte and to each other by intercellular bridges, called ring canals (Robinson et al., 1994). The syncytial ensemble of the 15 nurse cells and the oocyte is enveloped by an epithelial monolayer of somatic follicle cells and constitutes an ovarian follicle or an egg chamber, which is the structural and functional unit of the ovary (Spradling, 1993; Margaritis and Mazzini, 1998; Trougakos and Margaritis, 2002). The Drosophila ovary consists of a cluster of 18–20 ovarioles,
∗ Corresponding
author. Tel.: +30-10-727-4542; fax: +30-10-727-4742. E-mail address: [email protected] (L.H. Margaritis).
each one representing an independent, developmentally ordered follicle line. According to various morphogenetic criteria, the follicle development has been divided into multiple distinct stages (14 according to King, 1970; 20 according to Margaritis, 1985, 1986). During the late stages of oogenesis in D. melanogaster and in the olive fruit fly Dacus oleae, nurse cells die apoptotically (Cavaliere et al., 1998; Foley and Cooley, 1998; Nezis et al., 2000, 2001). Apoptosis or programmed cell death is an evolutionarily conserved, active and genetically regulated process, where cells that are no longer needed, are eliminated by activation of an intrinsic cell suicide program (Kerr et al., 1972; Steller, 1995). The execution of this cell death program is associated with characteristic morphological alterations, such as chromatin condensation, DNA fragmentation and reorganization of actin cytoskeleton (Kerr et al., 1972; Wyllie et al., 1980; Mills et al., 1999). The effector molecules of the apoptotic program are the cystein proteases, called caspases (Thornberry and Lazebnik, 1998). In the present study, we demonstrate the existence of two distinct apoptotic patterns in the nurse cells during oogenesis of the medfly Ceratitis capitata. One is developmentally regulated and normally occurs during stages 12 and 13, while the other is sporadically observed during stages 7 and 8. The
0040-8166/03/$ – see front matter © 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0040-8166(03)00010-7
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apoptotic manifestations of C. capitata nurse cells appear very similar to the ones occurring during D. melanogaster and D. oleae oogenesis (Nezis et al., 2000, 2001). These findings strongly suggest for a phylogenetically conserved mechanism during nurse cells apoptosis in higher Dipteran.
2. Materials and methods Ceratitis capitata (Diptera, Tephritidae) adult insects were kept in a 25 ◦ C culture room and slightly etherized before dissection. Dissections were carried out in cold Ringer’s solution and ovaries were separated into single ovarioles. Staging of the developing egg chambers during oogenesis was accomplished according to Margaritis (1986), Mouzaki and Margaritis (1991) and our observations. 2.1. Conventional light, transmission electron microscopy, phalloidin-FITC staining and TUNEL assay Ceratitis capitata egg chambers and whole ovaries were processed for conventional light, transmission electron microscopy, phalloidin-FITC and TUNEL labeling, as described elsewhere (Margaritis et al., 1980; Nezis et al., 2001, 2002). 2.2. Microscopy of the specimens Transmission electron microscopy preparations were examined using a Philips EM 300 operating at 60 kV. Slides for conventional light microscopy were examined with an Olympus BH-2 microscope. Confocal laser scanning microscopy was performed using a BioRad MRC 1024 laser-scanning confocal microscope.
3. Results 3.1. Nurse cells undergo apoptosis during the late stages of Ceratitis capitata oogenesis We used conventional light and transmission electron microscopy to detect the cellular events occurring during the
䉴 Fig. 1. Apoptotic events of the nurse cells during late oogenesis in Ceratitis capitata, as revealed by conventional light microscopy (resin sections stained with toluidine blue). (a)–(d) Light micrographs of C. capitata egg chambers, showing the anterior pole of the follicle. (a) Early stage 10: the nurse cell nuclei are euchromatic and almost spherical; O: oocyte, F: follicle cells, arrow: border cells. (b) Late stage 10: the nurse cell nuclei have retained the same morphology; NC: nurse cell, arrow: nurse cells nucleus. (c) Stage 11: some nurse cell nuclei show irregular shapes (arrows). (d) Stage 12: two nurse cell nuclei contain highly condensed chromatin and are disorganized (big arrows). The third one starts to condense (small arrow). Bars: (a) 100 m, (b)–(d) 10 m.
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late developmental stages of C. capitata oogenesis. At early stage 10, the nurse cells exhibit polygonal shape and their nuclei are almost spherical and euchromatic, containing distinct condensed masses that resemble nucleoli (Fig.1a). At late stage 10, the volume of the nurse cells has been reduced to half, since their cytoplasm has been pushed into the developing oocyte and their nuclei have been organized by the same structural pattern (Fig. 1b). During the following stage 11, the “dumping” process is completed and the nurse cells round up. Their nuclei remain mainly euchromatic, but some shape irregularities can also be detected (Fig. 1c). At stage 12, we can observe an asynchronous pattern of nuclear condensation, since the chromatin of some nurse cell nuclei starts to condense, while some others have already gained highly condensed chromatin (Fig. 1d). During stages 13 and 14, the chromatin of the nurse cell nuclei is highly condensed (Fig. 2d). The apoptotic nuclear remnants of the nurse cells are phagocytosed by the adjacent follicle cells and they can be observed within the follicle cell phagosomes (Fig. 2e). We also used TUNEL assay to demonstrate DNA fragmentation. At late stages 10 and 11, no signal is evident in the nurse cell cluster (Fig. 2a). The first positive signal can be detected at stage 12 (Fig. 2b). The same pattern can also be observed during the following stages 13 and 14 (Fig. 2c).
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3.2. Actin cytoskeleton reorganization during the late stages of Ceratitis capitata oogenesis We used phalloidin-FITC staining to detect filamentous actin network in C. capitata egg chambers. As illustrated in Fig. 3a, at the developmental stage 10 (10A), F-actin is localized subcortically in the nurse cells and is a major structural component of the ring canals. Stage 11 is characterized by a significant reorganization of actin cytoskeleton, since cytoplasmic actin bundles have been rearranged around the nurse cell nuclei (Fig. 3b). During stage 12, this actin network has been reorganized again and the major detectable cytoskeletal structure has been confined to multiple thick series of actin bundles traversing all the apoptotic nurse cells that have already obtained a round shape (Fig. 3c). Examination of the nurse cells cytoskeleton ultrastructure of stage 11 egg chambers revealed that actin filaments are assembled into actin bundles. These bundles overlap each other and form actin cables (Fig. 3d). During stage 12, the cables become very thick and traverse the disorganized apoptotic nurse cell nuclei (data not shown). It is important to emphasize that, the perinuclearly ordered actin arrangement, at stage 11, is remarkably converted to the non-symmetrically organized thick arrays of actin bundles,
Fig. 2. Confocal and electron micrographs demonstrating DNA fragmentation and chromatin condensation of nurse cell nuclei. (a)–(c) Confocal micrographs after TUNEL assay. (a) Late stage 10: no positive signal is evident at the nurse cell cluster (arrow). (b) Stage 12 and (c) stage 13: apoptotic nurse cell nuclei containing fragmented DNA (arrows). (d) Electron micrograph of stage 13 fragmented nurse cell nucleus containing highly condensed chromatin (N). (e) Electron micrograph of a stage 13 nurse cell, showing phagocytosis of the apoptotic nurse cell nuclear remnants by the adjacent follicle cells; NN: nurse cell nuclear remnants, FC: follicle cell, arrows: follicle cell phagosomes. Bars: (a)–(c) 100 m, (d) 10 m, (e) 1 m.
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death, strongly suggest that the ultrastructural changes of C. capitata actin network are triggered before stage 11, likely during stage 10B, as it has been clearly demonstrated for D. melanogaster (Nezis et al., 2000; Tilney et al., 1996; Riparbelli and Callaini, 1995). 3.3. Sporadic pattern of nurse cell apoptosis during mid-oogenesis In all the ovaries examined through light microscopy, we could observe a few abnormal egg chambers in low frequency (at least one per ovary). These egg chambers are exclusively restricted to developmental stages 7 and 8, and they are characterized by abnormal morphology compared to the normal egg chambers of the same stages (Fig. 4a and b). The follicular epithelium contains condensed apoptotic cell bodies, which are likely derived from the degenerated nurse cells and oocyte (Fig. 4a, c and d). Examination of the abnormal egg chamber ultrastructure revealed the presence of apoptotic cell bodies within the epithelial follicle cells (Fig. 4c and d). It is very likely that the nuclear and cytoplasmic remnants, originated from the apoptotic nurse cells and the oocyte, are effectively phagocytosed by the neighboring follicle cells. Interestingly, at these stages 7 and 8, all the surrounding follicle cells retain their ultrastructural integrity, with no detectable signals of apoptotic death.
4. Discussion
Fig. 3. Actin cytoskeleton reorganization during the late stages of oogenesis in Ceratitis capitata, as illustrated by phalloidin-FITC staining (a)–(c) and electron microscopy (d). (a)–(c) Confocal micrographs: (a) stage 10, (b) stage 11, and (c) stage 12. Arrows: (a) subcortical actin network; (b) perinuclearly organized cytoplasmic actin bundles; (c) thick actin cables traversing the nurse cells, arrowheads: ring canals; (d) electron micrograph of an actin cable (arrow) within a nurse cell of a stage 11 egg chamber. Note the stage-specific distinct patterns of F-actin network organization. Bars: (a)–(c) 100 m, (d) 1 m.
during following stages 12 and 13. The striking similarities of the cytoskeleton reorganization, between D. melanogaster (Nezis et al., 2000) and C. capitata nurse cells apoptotic
In the present study, we demonstrate two stage-specific patterns of programmed cell death during C. capitata oogenesis. The first one normally occurs during stages 12 and 13 of oogenesis. The second apoptotic pattern is sporadically observed during mid-oogenesis at stages 7 and 8. We have previously shown that in D. melanogaster and D. oleae oogenesis, nurse cells undergo apoptosis during stages 12 and 13, a process associated with actin cytoskeleton reorganization, chromatin condensation, DNA fragmentation and phagocytosis of the apoptotic nurse cell remnants by the adjacent follicle cells (Nezis et al., 2000, 2001). The physiological apoptotic pattern occurring during C. capitata oogenesis appears very similar to the one observed in D. melanogaster and remarkably similar to the one detected in D. oleae. The apoptotic pattern of the developmentally regulated nurse cell physiology can be divided into (a) the pre-apoptotic period of stages 10 and 11 and (b) the major apoptotic era of stages 12 and 13. The critical developmental stage 12 is characterized by TUNEL positive nuclei, reflecting fragmented nuclear DNA, while the initial observation of the actin cytoskeleton rearrangement is clearly detected during the pre-apoptotic stage 11. We strongly believe that the developmental changes of actin cytoskeleton organization, occurring during stages 10 and 11, are significantly implicated in both the nurse cells shaping and
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Fig. 4. Sporadic apoptotic pattern during mid-oogenesis in Ceratitis capitata. Light and electron micrographs of abnormal egg chambers during C. capitata oogenesis. (a) Light micrograph of an abnormal egg chamber stages 7 and 8. The oocyte and nurse cell cluster are completely degenerated. The follicular epithelium contains condensed apoptotic cell bodies (arrows). (b) Light micrograph of a normal stages 7 and 8 egg chamber. (c, d) Electron micrographs of abnormal egg chambers similar to (a). The apoptotic cell bodies are evident within the epithelial follicle cells (arrows); OC: oocyte, NC: nurse cells, FC: follicle cells, FN: follicle cell nucleus, AB: apoptotic body. Bars: (a, b) 50 m, (c) 10 m, (d) 5 m.
function, as well as in the physiological regulation of their apoptotic death. The pre-apoptotic manifestations begin at early stage 11 with the actin cytoskeleton reorganization, while the apoptotic ones continue with an asynchronous condensation of the nurse cell nuclei and DNA fragmentation at stage 12. They are finally terminated with the phagocytosis of the apoptotic remnants at stage 13 up to stage 14. This process is necessary for the normal maturation of the egg chamber. It is very likely that the massive depletion of apoptosis-inhibiting factors, due to the rapid transport of nurse cell cytoplasm into the oocyte, stimulates the apoptotic features that we have described, as it is demonstrated in D. melanogaster (Hay et al., 1995; Foley and Cooley, 1998; Nezis et al., 2000). The subcortical organization of actin filaments in nurse cells is altered to a perinuclear organization network during stages 10B and 11, and has been altered again during the apoptotic stages 12 and 13 to multiple thick series of actin cables traversing the nurse cells. The above cytoskeletal struc-
tural alterations consist essential apoptotic events, similar to the ones usually occurring in a large variety of cell types undergoing apoptosis (Kerr et al., 1972; Mills et al., 1999). Cytoskeleton-associated proteins, such as gelsolin, fodrin, Gas2 and FAK, may play critical roles in the cytoskeleton reorganization during apoptosis, since it has been previously reported that they can be specifically cleaved either by in vitro caspase treatment or after induction of apoptosis of the target cells in vivo (Martin et al., 1995; Kothakota et al., 1997; Wen et al., 1997; Janicke et al., 1998; Sgorbissa et al., 1999). In D. melanogaster more than five caspase-family members have been cloned and partially characterized so far with a representative example, the DCP-1 protein, which is directly implicated into the late developmental stages of oogenesis (Steller, 1995). The striking similarities of the apoptotic mechanisms, between D. melanogaster and C. capitata, allows us to strongly suggest that the genome of C. capitata contains a large number of caspase-family
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members, with significant homologies to their D. melanogaster counterparts (manuscript in preparation). We can speculate that the putative caspases of C. capitata could cleave and thereby alter the intracellular activity of actin binding proteins, as it has been previously reported for the caspase-3 mammalian homologue, which can specifically cleave and activate the gelsolin cytoskeletal component during apoptotic stimulation (Kothakota et al., 1997). The regulation and implementation of this cell death program needs further clarification at both the cellular and molecular level. The second apoptotic pattern occurs sporadically during the developmental stages 7 and 8 in low frequency. A similar apoptotic process has been previously described in D. melanogaster (Giorgi and Deri, 1976; Nezis et al., 2000) and also observed in D. oleae (Nezis and Margaritis, unpublished data). The availability of food and other environmental factors can influence the ability of D. melanogaster egg chambers to develop beyond stages 7 and 8 (Spradling, 1993; Drummond-Barbosa and Spradling, 2001). Also, disruption of the follicle cell layer induces germline cell death during mid-oogenesis in D. melanogaster (De Lorenzo et al., 1999; Chao and Nagoshi, 1999). Furthermore, treatment of D. melanogaster egg chambers with the apoptotic inducers etoposide and staurosporine triggers nurse cells apoptosis during stages 7 and 8 (Nezis et al., 2000). It has also been suggested that ecdysone, produced by nurse cells, is required through an autocrine mechanism, by the germline cells for egg chamber physiological maturation during mid-oogenesis in D. melanogaster (Buszczak et al., 1999). It is likely that a similar mechanism might exist in C. capitata too. When an egg chamber enters vitellogenesis during stages 7 and 8, it needs significant amount of resources. Therefore, previtellogenic “control mechanisms” may serve to eliminate defective egg chambers unable to produce viable progeny and thus may prevent the waste of precious nutrients. This observation suggests that mid-oogenesis in C. capitata may be the critical regulation point at which defective or abnormal egg chambers are eliminated. Thus, it is likely that this phenomenon consists of a protective mechanism throughout C. capitata oogenesis, when damaged or abnormal follicles enter apoptosis before they reach maturity. The two stage-specific apoptotic patterns observed in C. capitata are remarkably similar to the ones occurring during D. oleae oogenesis (Nezis et al., 2001; Nezis and Margaritis, unpublished data). All the above observations justify the inclusion of these two insect species in the same taxonomical group, as it has been demonstrated before by a large variety of different criteria (Kristensen, 1981). Also D. melanogaster, another higher Dipteran, exhibits similar developmentally regulated apoptotic patterns (Nezis et al., 2000). These findings strongly suggest for a phylogenetically conserved mechanism of nurse cells apoptosis, which plays an essential role in normal oogenesis of higher Dipteran.
Acknowledgements The authors would like to thank Dr. S. Loukas (Institute of Biology, National Center for Scientific Research “Demokritos”, Athens, Greece) for kindly providing CLSM facilities. This work was supported by grant to L.H. Margaritis from European Union (TMR-Network, Grant No. ERBFMRXCT 980200). References Buszczak, M., Freeman, M.R., Carlson, J.R., Bender, M., Cooley, L., Segraves, W.A., 1999. Ecdysone response genes govern egg chamber development during mid-oogenesis in Drosophila. Development 126, 4581–4589. Cavaliere, V., Taddei, C., Gargiulo, G., 1998. Apoptosis of nurse cells at the late stages of oogenesis of Drosophila melanogaster. Dev. Genes Evol. 208, 106–112. Chao, S., Nagoshi, R.N., 1999. Induction of apoptosis in the germline and follicle layer of Drosophila egg chambers. Mech. Dev. 88, 159–172. De Lorenzo, C., Strand, D., Mechler, B.M., 1999. Requirement of Drosophila l(2)gl function for survival of the germline cells and organization of the follicle cells in a columnar epithelium during oogenesis. Int. J. Dev. Biol. 43, 207–217. Drummond-Barbosa, D., Spradling, A.C., 2001. Stem cells and their progeny respond to nutritional changes during Drosophila oogenesis. Dev. Biol. 231, 265–278. Foley, K., Cooley, L., 1998. Apoptosis in late stage Drosophila nurse cells does not require genes within the H99 deficiency. Development 125, 1075–1082. Giorgi, F., Deri, P., 1976. Cell death in ovarian chambers of Drosophila melanogaster. J. Embryol. Exp. Morphol. 35, 521–533. Gutzeit, H.O., 1986. Transport of molecules and organelles in meroistic ovarioles of insects. Differentiation 31, 155–165. Hay, B.A., Wassarman, D.A., Rubin, G.M., 1995. Drosophila homologs of baculovirus inhibitor of apoptosis proteins function to block cell death. Cell 83, 1253–1262. Janicke, R.U., Ng, P., Sprengart, M.L., Porter, A.G., 1998. Caspase-3 is required for alpha-fodrin cleavage but dispensable for cleavage of other death substrates in apoptosis. J. Biol. Chem. 273, 15540– 15545. Kerr, J.F.R., Wyllie, A.H., Currie, A.R., 1972. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer 26, 239–257. King, R.C., 1970. Origin and development of the egg chamber within the adult ovarioles. In: Ovarian Development in Drosophila melanogaster. Academic Press, New York/London, pp. 38–54. Kothakota, S., Azuma, T., Reinhard, C., Klippel, A., Tang, J., Chu, K., McGarry, T.J., Kirschner, M.W., Koths, K., Kwiatkowski, D.J., Williams, L.T., 1997. Caspase-3-generated fragment of gelsolin: effector of morphological change in apoptosis. Science 278, 294– 298. Kristensen, N.P., 1981. Phylogeny of insect orders. Ann. Rev. Entomol. 26, 135–157. Margaritis, L.H., 1985. Structure and physiology of the eggshell. In: Gilbert, L.I., Kerkut, G.A. (Eds.), Comprehensive Insect Biochemistry, Physiology and Pharmacology, vol. 1. Pergamon Press, Oxford, New York, pp. 151–230. Margaritis, L.H., 1986. The eggshell of Drosophilla melanogaster. New staging characteristics and fine structural analysis of choriogenesis. Can. J. Zool. 64, 2152–2175. Margaritis, L.H., Mazzini, M., 1998. Structure of the egg. In: Harrison, F.W., Locke, M. (Eds.), Microscopic Anatomy of Invertebrates. Vol. 11C “Insecta”. Wiley and Liss, New York, pp. 995–1037.
I.P. Nezis et al. / Tissue & Cell 35 (2003) 113–119 Margaritis, L.H., Kafatos, F.C., Petri, W.H., 1980. The eggshell of Drosophila melanogaster. I. The fine structure of the layers and regions of the wild type eggshell. J. Cell. Sci. 43, 1–35. Martin, S.J., O’Brien, G.A., Nishioka, W.K., McGahon, A.J., Mahboudi, A., Saido, T.C., Green, D.R., 1995. Proteolysis of fodrin (non-erythroid spectrin) during apoptosis. J. Biol. Chem. 270, 6425–6428. Mills, J.C., Stone, N.L., Pittman, R.N., 1999. Extranuclear apoptosis. The role of the cytoplasm in the execution phase. J. Cell. Biol. 146, 703– 708. Mouzaki, D.G., Margaritis, L.H., 1991. Choriogenesis in the medfly Ceratitis capitata (Wiedermann) (Diptera: Tephritidae). Int. J. Insect Morphol. 20, 51–68. Nezis, I.P., Stravopodis, D.J., Papassideri, I., Robert-Nicoud, M., Margaritis, L.H., 2000. Stage-specific apoptotic patterns during Drosophila oogenesis. Eur. J. Cell Biol. 79, 610–620. Nezis, I.P., Stravopodis, D.J., Papassideri, I., Margaritis, L.H., 2001. Actin cytoskeleton reorganization of the apoptotic nurse cells during the late developmental stages of oogenesis in Dacus oleae. Cell Motil. Cytoskeleton 48, 224–233. Nezis, I.P., Stravopodis, D.J., Papassideri, I., Robert-Nicoud, M., Margaritis, L.H., 2002. The dynamics of apoptosis in the ovarian follicle cells during the late stages of Drosophila oogenesis. Cell Tissue Res. 307, 401–409. Riparbelli, M.G., Callaini, G., 1995. Cytoskeleton of the Drosophila egg chamber: new observations on microfilament distribution during oocyte growth. Cell Motil. Cytoskeleton 31, 298–306.
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Robinson, D.N., Kant, K., Cooley, L., 1994. Morphogenesis of Drosophila ovarian ring canals. Development 120, 2015–2025. Sgorbissa, A., Benetti, R., Marzinotto, S., Schneider, C., Brancolini, C., 1999. Caspase-3 and caspase-7 but not caspase-6 cleave Gas2 in vitro: implications for microfilament reorganization during apoptosis. J. Cell Sci. 112, 4475–4482. Spradling, A.C., 1993. Developmental genetics of oogenesis. In: Bate, M., Martinez-Arias, A. (Eds.), The Development of Drosophila melanogaster, vol. 1. Cold Spring Harbor Press, Cold Spring Harbor, NY, pp. 1–70. Steller, H., 1995. Mechanisms and genes of cellular suicide. Science 267, 1445–1449. Thornberry, N.A., Lazebnik, Y., 1998. Caspases: enemies within. Science 28, 281 (5381), 1312–1316. Tilney, L.G., Connelly, P., Smith, S., Guild, G.M., 1996. F-actin bundles in Drosophila bristles are assembled from modules composed of short filaments. J. Cell Biol. 135, 1291–1308. Trougakos, I.P., Margaritis, L.H., 2002. Novel morphological and physiological aspects of insect eggs. In: Hilker, M., Meiners, T. (Eds.), Chemoecology of Insect Eggs and Egg Deposition. Blackwell Wissenschaftsverlag, Berlin, Germany, pp. 3–36. Wen, L.P., Fahrni, J.A., Troie, S., Guan, J.L., Orth, K., Rosen, G.D., 1997. Cleavage of focal adhesion kinase by caspases during apoptosis. J. Biol. Chem. 272, 26056–26061. Wyllie, A.H., Kerr, J.F.R., Currie, A.R., 1980. Cell death: the significance of apoptosis. Int. Rev. Cytol. 68, 251–306.
Modes of programmed cell death during Ceratitis capitata oogenesis Ioannis P. Nezis a , Vassilis Modes a , Vicky Mpakou a , Dimitrios J. Stravopodis a , Issidora S. Papassideri a , Ioanna Mammali b , Lukas H. Margaritis a,∗ a
Faculty of Biology, Department of Cell Biology and Biophysics, University of Athens, Panepistimiopolis 15784, Athens, Greece b Department of Neurology, Aiginition Hospital, University of Athens, Panepistimiopolis 15784, Athens, Greece Received 7 October 2002; received in revised form 27 December 2002; accepted 27 December 2002
Abstract In the present study, we demonstrate the existence of two distinct apoptotic patterns in nurse cells during Ceratitis capitata oogenesis. One is developmentally regulated and normally occurs during stages 12 and 13, and the other is stage specific and is sporadically observed during stages 7 and 8. The pre-apoptotic manifestation of the first pattern begins at stage 11 and is characterized by the formation of actin bundles. Subsequently, at stages 12 and 13, the nurse cell nuclei exhibit condensed chromatin and contain fragmented DNA, as revealed by TUNEL assay. The apoptotic nurse cell remnants are phagocytosed by the neighboring follicle cells at the end of oogenesis during stages 13 and 14. In the second apoptotic pattern, which occurs sporadically during stages 7 and 8, the nurse cells degenerate and are phagocytosed by the follicular epithelium that contains apoptotic cell bodies. The data presented herein, compared to previous reported results in Drosophila melanogaster and Dacus oleae (Nezis et al., 2000, 2001), strongly suggest that nurse cell apoptosis is a developmentally regulated and phylogenetically conserved mechanism in higher Dipteran. They also suggest that, the sporadic apoptotic pattern consists of a possible protective mechanism throughout oogenesis when damaged or abnormal egg chambers, are eliminated before they reach maturity. © 2003 Elsevier Science Ltd. All rights reserved. Keywords: Actin cytoskeleton; Apoptosis; Diptera; Oogenesis; Phagocytosis
1. Introduction Oogenesis is a significant biological process that leads to the formation of a highly complex cell, the oocyte. One of its major characteristics is the remarkable gradual increase in the volume of the oocyte due to accumulation of nutrients, mRNAs, proteins and organelles required during early embryonic development. In insects, the acquisition of the above nutritious material can be achieved by several different ways (Gutzeit, 1986). In some insects, including Drosophila melanogaster, the oocyte development is supported by a group of cells, termed nurse cells, which are connected to the oocyte and to each other by intercellular bridges, called ring canals (Robinson et al., 1994). The syncytial ensemble of the 15 nurse cells and the oocyte is enveloped by an epithelial monolayer of somatic follicle cells and constitutes an ovarian follicle or an egg chamber, which is the structural and functional unit of the ovary (Spradling, 1993; Margaritis and Mazzini, 1998; Trougakos and Margaritis, 2002). The Drosophila ovary consists of a cluster of 18–20 ovarioles,
∗ Corresponding
author. Tel.: +30-10-727-4542; fax: +30-10-727-4742. E-mail address: [email protected] (L.H. Margaritis).
each one representing an independent, developmentally ordered follicle line. According to various morphogenetic criteria, the follicle development has been divided into multiple distinct stages (14 according to King, 1970; 20 according to Margaritis, 1985, 1986). During the late stages of oogenesis in D. melanogaster and in the olive fruit fly Dacus oleae, nurse cells die apoptotically (Cavaliere et al., 1998; Foley and Cooley, 1998; Nezis et al., 2000, 2001). Apoptosis or programmed cell death is an evolutionarily conserved, active and genetically regulated process, where cells that are no longer needed, are eliminated by activation of an intrinsic cell suicide program (Kerr et al., 1972; Steller, 1995). The execution of this cell death program is associated with characteristic morphological alterations, such as chromatin condensation, DNA fragmentation and reorganization of actin cytoskeleton (Kerr et al., 1972; Wyllie et al., 1980; Mills et al., 1999). The effector molecules of the apoptotic program are the cystein proteases, called caspases (Thornberry and Lazebnik, 1998). In the present study, we demonstrate the existence of two distinct apoptotic patterns in the nurse cells during oogenesis of the medfly Ceratitis capitata. One is developmentally regulated and normally occurs during stages 12 and 13, while the other is sporadically observed during stages 7 and 8. The
0040-8166/03/$ – see front matter © 2003 Elsevier Science Ltd. All rights reserved. doi:10.1016/S0040-8166(03)00010-7
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apoptotic manifestations of C. capitata nurse cells appear very similar to the ones occurring during D. melanogaster and D. oleae oogenesis (Nezis et al., 2000, 2001). These findings strongly suggest for a phylogenetically conserved mechanism during nurse cells apoptosis in higher Dipteran.
2. Materials and methods Ceratitis capitata (Diptera, Tephritidae) adult insects were kept in a 25 ◦ C culture room and slightly etherized before dissection. Dissections were carried out in cold Ringer’s solution and ovaries were separated into single ovarioles. Staging of the developing egg chambers during oogenesis was accomplished according to Margaritis (1986), Mouzaki and Margaritis (1991) and our observations. 2.1. Conventional light, transmission electron microscopy, phalloidin-FITC staining and TUNEL assay Ceratitis capitata egg chambers and whole ovaries were processed for conventional light, transmission electron microscopy, phalloidin-FITC and TUNEL labeling, as described elsewhere (Margaritis et al., 1980; Nezis et al., 2001, 2002). 2.2. Microscopy of the specimens Transmission electron microscopy preparations were examined using a Philips EM 300 operating at 60 kV. Slides for conventional light microscopy were examined with an Olympus BH-2 microscope. Confocal laser scanning microscopy was performed using a BioRad MRC 1024 laser-scanning confocal microscope.
3. Results 3.1. Nurse cells undergo apoptosis during the late stages of Ceratitis capitata oogenesis We used conventional light and transmission electron microscopy to detect the cellular events occurring during the
䉴 Fig. 1. Apoptotic events of the nurse cells during late oogenesis in Ceratitis capitata, as revealed by conventional light microscopy (resin sections stained with toluidine blue). (a)–(d) Light micrographs of C. capitata egg chambers, showing the anterior pole of the follicle. (a) Early stage 10: the nurse cell nuclei are euchromatic and almost spherical; O: oocyte, F: follicle cells, arrow: border cells. (b) Late stage 10: the nurse cell nuclei have retained the same morphology; NC: nurse cell, arrow: nurse cells nucleus. (c) Stage 11: some nurse cell nuclei show irregular shapes (arrows). (d) Stage 12: two nurse cell nuclei contain highly condensed chromatin and are disorganized (big arrows). The third one starts to condense (small arrow). Bars: (a) 100 m, (b)–(d) 10 m.
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late developmental stages of C. capitata oogenesis. At early stage 10, the nurse cells exhibit polygonal shape and their nuclei are almost spherical and euchromatic, containing distinct condensed masses that resemble nucleoli (Fig.1a). At late stage 10, the volume of the nurse cells has been reduced to half, since their cytoplasm has been pushed into the developing oocyte and their nuclei have been organized by the same structural pattern (Fig. 1b). During the following stage 11, the “dumping” process is completed and the nurse cells round up. Their nuclei remain mainly euchromatic, but some shape irregularities can also be detected (Fig. 1c). At stage 12, we can observe an asynchronous pattern of nuclear condensation, since the chromatin of some nurse cell nuclei starts to condense, while some others have already gained highly condensed chromatin (Fig. 1d). During stages 13 and 14, the chromatin of the nurse cell nuclei is highly condensed (Fig. 2d). The apoptotic nuclear remnants of the nurse cells are phagocytosed by the adjacent follicle cells and they can be observed within the follicle cell phagosomes (Fig. 2e). We also used TUNEL assay to demonstrate DNA fragmentation. At late stages 10 and 11, no signal is evident in the nurse cell cluster (Fig. 2a). The first positive signal can be detected at stage 12 (Fig. 2b). The same pattern can also be observed during the following stages 13 and 14 (Fig. 2c).
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3.2. Actin cytoskeleton reorganization during the late stages of Ceratitis capitata oogenesis We used phalloidin-FITC staining to detect filamentous actin network in C. capitata egg chambers. As illustrated in Fig. 3a, at the developmental stage 10 (10A), F-actin is localized subcortically in the nurse cells and is a major structural component of the ring canals. Stage 11 is characterized by a significant reorganization of actin cytoskeleton, since cytoplasmic actin bundles have been rearranged around the nurse cell nuclei (Fig. 3b). During stage 12, this actin network has been reorganized again and the major detectable cytoskeletal structure has been confined to multiple thick series of actin bundles traversing all the apoptotic nurse cells that have already obtained a round shape (Fig. 3c). Examination of the nurse cells cytoskeleton ultrastructure of stage 11 egg chambers revealed that actin filaments are assembled into actin bundles. These bundles overlap each other and form actin cables (Fig. 3d). During stage 12, the cables become very thick and traverse the disorganized apoptotic nurse cell nuclei (data not shown). It is important to emphasize that, the perinuclearly ordered actin arrangement, at stage 11, is remarkably converted to the non-symmetrically organized thick arrays of actin bundles,
Fig. 2. Confocal and electron micrographs demonstrating DNA fragmentation and chromatin condensation of nurse cell nuclei. (a)–(c) Confocal micrographs after TUNEL assay. (a) Late stage 10: no positive signal is evident at the nurse cell cluster (arrow). (b) Stage 12 and (c) stage 13: apoptotic nurse cell nuclei containing fragmented DNA (arrows). (d) Electron micrograph of stage 13 fragmented nurse cell nucleus containing highly condensed chromatin (N). (e) Electron micrograph of a stage 13 nurse cell, showing phagocytosis of the apoptotic nurse cell nuclear remnants by the adjacent follicle cells; NN: nurse cell nuclear remnants, FC: follicle cell, arrows: follicle cell phagosomes. Bars: (a)–(c) 100 m, (d) 10 m, (e) 1 m.
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death, strongly suggest that the ultrastructural changes of C. capitata actin network are triggered before stage 11, likely during stage 10B, as it has been clearly demonstrated for D. melanogaster (Nezis et al., 2000; Tilney et al., 1996; Riparbelli and Callaini, 1995). 3.3. Sporadic pattern of nurse cell apoptosis during mid-oogenesis In all the ovaries examined through light microscopy, we could observe a few abnormal egg chambers in low frequency (at least one per ovary). These egg chambers are exclusively restricted to developmental stages 7 and 8, and they are characterized by abnormal morphology compared to the normal egg chambers of the same stages (Fig. 4a and b). The follicular epithelium contains condensed apoptotic cell bodies, which are likely derived from the degenerated nurse cells and oocyte (Fig. 4a, c and d). Examination of the abnormal egg chamber ultrastructure revealed the presence of apoptotic cell bodies within the epithelial follicle cells (Fig. 4c and d). It is very likely that the nuclear and cytoplasmic remnants, originated from the apoptotic nurse cells and the oocyte, are effectively phagocytosed by the neighboring follicle cells. Interestingly, at these stages 7 and 8, all the surrounding follicle cells retain their ultrastructural integrity, with no detectable signals of apoptotic death.
4. Discussion
Fig. 3. Actin cytoskeleton reorganization during the late stages of oogenesis in Ceratitis capitata, as illustrated by phalloidin-FITC staining (a)–(c) and electron microscopy (d). (a)–(c) Confocal micrographs: (a) stage 10, (b) stage 11, and (c) stage 12. Arrows: (a) subcortical actin network; (b) perinuclearly organized cytoplasmic actin bundles; (c) thick actin cables traversing the nurse cells, arrowheads: ring canals; (d) electron micrograph of an actin cable (arrow) within a nurse cell of a stage 11 egg chamber. Note the stage-specific distinct patterns of F-actin network organization. Bars: (a)–(c) 100 m, (d) 1 m.
during following stages 12 and 13. The striking similarities of the cytoskeleton reorganization, between D. melanogaster (Nezis et al., 2000) and C. capitata nurse cells apoptotic
In the present study, we demonstrate two stage-specific patterns of programmed cell death during C. capitata oogenesis. The first one normally occurs during stages 12 and 13 of oogenesis. The second apoptotic pattern is sporadically observed during mid-oogenesis at stages 7 and 8. We have previously shown that in D. melanogaster and D. oleae oogenesis, nurse cells undergo apoptosis during stages 12 and 13, a process associated with actin cytoskeleton reorganization, chromatin condensation, DNA fragmentation and phagocytosis of the apoptotic nurse cell remnants by the adjacent follicle cells (Nezis et al., 2000, 2001). The physiological apoptotic pattern occurring during C. capitata oogenesis appears very similar to the one observed in D. melanogaster and remarkably similar to the one detected in D. oleae. The apoptotic pattern of the developmentally regulated nurse cell physiology can be divided into (a) the pre-apoptotic period of stages 10 and 11 and (b) the major apoptotic era of stages 12 and 13. The critical developmental stage 12 is characterized by TUNEL positive nuclei, reflecting fragmented nuclear DNA, while the initial observation of the actin cytoskeleton rearrangement is clearly detected during the pre-apoptotic stage 11. We strongly believe that the developmental changes of actin cytoskeleton organization, occurring during stages 10 and 11, are significantly implicated in both the nurse cells shaping and
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Fig. 4. Sporadic apoptotic pattern during mid-oogenesis in Ceratitis capitata. Light and electron micrographs of abnormal egg chambers during C. capitata oogenesis. (a) Light micrograph of an abnormal egg chamber stages 7 and 8. The oocyte and nurse cell cluster are completely degenerated. The follicular epithelium contains condensed apoptotic cell bodies (arrows). (b) Light micrograph of a normal stages 7 and 8 egg chamber. (c, d) Electron micrographs of abnormal egg chambers similar to (a). The apoptotic cell bodies are evident within the epithelial follicle cells (arrows); OC: oocyte, NC: nurse cells, FC: follicle cells, FN: follicle cell nucleus, AB: apoptotic body. Bars: (a, b) 50 m, (c) 10 m, (d) 5 m.
function, as well as in the physiological regulation of their apoptotic death. The pre-apoptotic manifestations begin at early stage 11 with the actin cytoskeleton reorganization, while the apoptotic ones continue with an asynchronous condensation of the nurse cell nuclei and DNA fragmentation at stage 12. They are finally terminated with the phagocytosis of the apoptotic remnants at stage 13 up to stage 14. This process is necessary for the normal maturation of the egg chamber. It is very likely that the massive depletion of apoptosis-inhibiting factors, due to the rapid transport of nurse cell cytoplasm into the oocyte, stimulates the apoptotic features that we have described, as it is demonstrated in D. melanogaster (Hay et al., 1995; Foley and Cooley, 1998; Nezis et al., 2000). The subcortical organization of actin filaments in nurse cells is altered to a perinuclear organization network during stages 10B and 11, and has been altered again during the apoptotic stages 12 and 13 to multiple thick series of actin cables traversing the nurse cells. The above cytoskeletal struc-
tural alterations consist essential apoptotic events, similar to the ones usually occurring in a large variety of cell types undergoing apoptosis (Kerr et al., 1972; Mills et al., 1999). Cytoskeleton-associated proteins, such as gelsolin, fodrin, Gas2 and FAK, may play critical roles in the cytoskeleton reorganization during apoptosis, since it has been previously reported that they can be specifically cleaved either by in vitro caspase treatment or after induction of apoptosis of the target cells in vivo (Martin et al., 1995; Kothakota et al., 1997; Wen et al., 1997; Janicke et al., 1998; Sgorbissa et al., 1999). In D. melanogaster more than five caspase-family members have been cloned and partially characterized so far with a representative example, the DCP-1 protein, which is directly implicated into the late developmental stages of oogenesis (Steller, 1995). The striking similarities of the apoptotic mechanisms, between D. melanogaster and C. capitata, allows us to strongly suggest that the genome of C. capitata contains a large number of caspase-family
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members, with significant homologies to their D. melanogaster counterparts (manuscript in preparation). We can speculate that the putative caspases of C. capitata could cleave and thereby alter the intracellular activity of actin binding proteins, as it has been previously reported for the caspase-3 mammalian homologue, which can specifically cleave and activate the gelsolin cytoskeletal component during apoptotic stimulation (Kothakota et al., 1997). The regulation and implementation of this cell death program needs further clarification at both the cellular and molecular level. The second apoptotic pattern occurs sporadically during the developmental stages 7 and 8 in low frequency. A similar apoptotic process has been previously described in D. melanogaster (Giorgi and Deri, 1976; Nezis et al., 2000) and also observed in D. oleae (Nezis and Margaritis, unpublished data). The availability of food and other environmental factors can influence the ability of D. melanogaster egg chambers to develop beyond stages 7 and 8 (Spradling, 1993; Drummond-Barbosa and Spradling, 2001). Also, disruption of the follicle cell layer induces germline cell death during mid-oogenesis in D. melanogaster (De Lorenzo et al., 1999; Chao and Nagoshi, 1999). Furthermore, treatment of D. melanogaster egg chambers with the apoptotic inducers etoposide and staurosporine triggers nurse cells apoptosis during stages 7 and 8 (Nezis et al., 2000). It has also been suggested that ecdysone, produced by nurse cells, is required through an autocrine mechanism, by the germline cells for egg chamber physiological maturation during mid-oogenesis in D. melanogaster (Buszczak et al., 1999). It is likely that a similar mechanism might exist in C. capitata too. When an egg chamber enters vitellogenesis during stages 7 and 8, it needs significant amount of resources. Therefore, previtellogenic “control mechanisms” may serve to eliminate defective egg chambers unable to produce viable progeny and thus may prevent the waste of precious nutrients. This observation suggests that mid-oogenesis in C. capitata may be the critical regulation point at which defective or abnormal egg chambers are eliminated. Thus, it is likely that this phenomenon consists of a protective mechanism throughout C. capitata oogenesis, when damaged or abnormal follicles enter apoptosis before they reach maturity. The two stage-specific apoptotic patterns observed in C. capitata are remarkably similar to the ones occurring during D. oleae oogenesis (Nezis et al., 2001; Nezis and Margaritis, unpublished data). All the above observations justify the inclusion of these two insect species in the same taxonomical group, as it has been demonstrated before by a large variety of different criteria (Kristensen, 1981). Also D. melanogaster, another higher Dipteran, exhibits similar developmentally regulated apoptotic patterns (Nezis et al., 2000). These findings strongly suggest for a phylogenetically conserved mechanism of nurse cells apoptosis, which plays an essential role in normal oogenesis of higher Dipteran.
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