An in vitro analysis of ecdysteroid-elicited cell death in the prothoracic gland of Manduca sexta

The prothoracic glands of the tobacco hornworm, Manduca sexta, secrete the precursor of the insect molting hormone and normally undergo programmed cell death (PCD) during pupal-adult metamorphosis, between days 5 and 6 after pupation. This phenomenon can be elicited prematurely in vitro by the addition of 20-hydroxyecdysone (20E) to the gland cultures. To induce nuclear condensation in vitro in the glands from day-1 pupae, the effective dose range of 20E is 0.7-7 micrograms/ml and the minimum exposure period is 24 h. Prothoracic glands from different stages of pupal-adult development express different responsiveness to exogenous ecdysteroids. By utilizing terminal deoxynucleotidyl-transferase-mediated dUTP nick-end-labeling (TUNEL) and the apoptotic DNA laddering method together with transmission electron microscopy, it has been demonstrated that the ecdysteroid-induced cell death of the prothoracic glands occurs via not only apoptosis but also autophagy, i.e., the induced dying cells show both severe nuclear fragmentation and autophagic vacuole formation, characteristics typical of apoptotic and autophagic cell death. The composite data indicate that ecdysteroids regulate directly both apoptotic and autophagic mechanisms of PCD of the prothoracic glands.     

Programmed cell death in Bradysia hygida (Diptera, Sciaridae) salivary glands presents apoptotic features

In this work, we present biochemical and morphological evidence that the final steps of programmed cell death (PCD) in the salivary glands of the inferior Diptera, Bradysia hygida, present apoptotic characteristics. In B. hygida, elimination of salivary glands is preceded by the establishment of a typical pattern of protein synthesis; increase in caspase activity; decrease in cell volume; nuclear pyknosis; nuclear DNA breakage; changes in the actin cytoskeleton; and most importantly, destruction of giant cells via formation of apoptotic bodies containing broken DNA or cytoplasm remains. Thus, elimination of B. hygida salivary glands by this process suggests that such mode of PCD is also involved in the destruction of entire organs in insects and, therefore, adds more complexity to the regulation of tissue elimination during development.     

Caspases function in autophagic programmed cell death in Drosophila

Self-digestion of cytoplasmic components is the hallmark of autophagic programmed cell death. This auto-degradation appears to be distinct from what occurs in apoptotic cells that are engulfed and digested by phagocytes. Although much is known about apoptosis, far less is known about the mechanisms that regulate autophagic cell death. Here we show that autophagic cell death is regulated by steroid activation of caspases in Drosophila salivary glands. Salivary glands exhibit some morphological changes that are similar to apoptotic cells, including fragmentation of the cytoplasm, but do not appear to use phagocytes in their degradation. Changes in the levels and localization of filamentous Actin, alpha-Tubulin, alpha-Spectrin and nuclear Lamins precede salivary gland destruction, and coincide with increased levels of active Caspase 3 and a cleaved form of nuclear Lamin. Mutations in the steroid-regulated genes beta FTZ-F1, E93, BR-C and E74A that prevent salivary gland cell death possess altered levels and localization of filamentous Actin, alpha-Tubulin, alpha-Spectrin, nuclear Lamins and active Caspase 3. Inhibition of caspases, by expression of either the caspase inhibitor p35 or a dominant-negative form of the initiator caspase Dronc, is sufficient to inhibit salivary gland cell death, and prevent changes in nuclear Lamins and alpha-Tubulin, but not to prevent the reorganization of filamentous Actin. These studies suggest that aspects of the cytoskeleton may be required for changes in dying salivary glands. Furthermore, caspases are not only used during apoptosis, but also function in the regulation of autophagic cell death.     

Real-time observation of autophagic programmed cell death of<Emphasis Type="Italic"> Drosophila</Emphasis> salivary glands in vitro

Autophagy, a form of programmed cell death (PCD) that is morphologically distinguished from apoptosis, is thought to be as prevalent as apoptosis, at least during development. In insect metamorphosis, the steroid hormone 20-hydroxyecdysone (ecdysone) activates autophagic PCD to eliminate larval structures that are no longer needed. However, in comparison with apoptosis, there are not many studies on the regulation mechanisms of autophagy. To provide a useful model for studying autophagic PCD, I established an in vitro culture system that enables real-time observation of the autophagic cell destruction of Drosophila salivary glands. The new system revealed that de novo gene expression was still required for the destruction of salivary glands dissected from phanerocephalic pupae. This indicates the usefulness of the system for exploring genes that participate in the last processes of autophagic PCD.Edited by N. Satoh     

Drosophila salivary gland proteins and pupation

Pulse-labeling experiments of salivary glands from the prepupal stages of development showed selectively high rates of synthesis of a set of low molecular weight proteins (6K–12K). These proteins are stably maintained in the salivary glands during prepupal development and are subsequently transported to the pupation fluid (found between the pupal case and the prepupal cuticle) when pupation occurs. These small polypeptides are very basic with the major components having isoelectric points of 8.6–8.7 and the minor components having isoelectric points of 9.1–9.5. This study shows the continuing function of the salivary glands—specifically, the synthesis and secretion of a set of proteins with a putative role in pupation.     

AP-1, but not NF-kappa B, is required for efficient steroid-triggered cell death in Drosophila

Extensive studies in vertebrate cells have assigned a central role to Rel/NF-kappa B and AP-1 family members in the control of apoptosis. We ask here whether parallel pathways might function in Drosophila by determining if Rel/NF-kappa B or AP-1 family members contribute to the steroid-triggered death of larval salivary glands during Drosophila metamorphosis. We show that two of the three Drosophila Rel/NF-kappa B genes are expressed in doomed salivary glands and that one family member, Dif, is induced in a stage-specific manner immediately before the onset of programmed cell death. Similarly, Djun is expressed for many hours before salivary gland cell death while Dfos is induced in a stage-specific manner, immediately before this tissue is destroyed. We show that null mutations in the three Drosophila Rel/NF-kappa B family members, either alone or in combination, have no apparent effect on this death response. In contrast, Dfos is required for the proper timing of larval salivary gland cell death as well as the proper induction of key death genes. This study demonstrates a role for AP-1 in the stage-specific steroid-triggered programmed cell death of larval tissues during Drosophila metamorphosis.     

Programmed cell death of larval tissues induced by juvenile hormone in the bamboo borer, Omphisa fuscidentalis

Programmed cell death (PCD) plays a critical role during animal development through the destruction of unneeded cells and tissues. In some insects, the prothoracic glands (PGs) and anterior silk glands (ASGs) are larval-specific tissues that are normally eliminated by PCD after pupation. Previous studies report that juvenile hormone analog (JHA) terminates the larval diapause of Omphisa fuscidentalis by increasing the hemolymph ecdysteroids that trigger PCD. Because JHA may indirectly induce the PCD of the PGs and ASGs of Omphisa diapausing larvae, the effects of JHA on the induction of PCD were determined. The application of 1μg JHA induced PCD in the PGs and ASGs of larvae identified as stage G0 (prior to pupation). The injection of 1μg 20E triggered the PCD of the ASGs when the larvae expressed a G0-G1 morphology, whereas PCD occurred in the PGs on day 1 post-injection. Histological studies revealed similar patterns of morphological changes during the PG and ASG PCD in the JHA- and 20E-treated larvae. Furthermore, to confirm that PCD was induced by a high ecdysteroid level that increases after JHA application, the expression profiles of EcR-A and EcR-B1 in the PGs and ASGs from the JHA-treated larvae were examined, and the results showed that the expression levels of EcR-A and EcR-B1 mRNA increased during the G0 stage. These results suggest that JHA may be involved in PCD by increasing the ecdysteroid titer, leading to termination of the larval diapause period in Omphisa fuscidentalis.     

Programmed cell death in the ascidian embryo: modulation by FoxA5 and Manx and roles in the evolution of larval development

Programmed cell death (PCD) has been discounted in the ascidian embryo because the descendants of every embryonic cell appear to be present in the tadpole larva. Here we show that apoptotic PCD is initiated in the epidermis and central nervous system (CNS) but not in the endoderm, mesenchyme, muscle, and notochord cells during embryogenesis in molgulid ascidians. However, the affected cells do not actually die until the beginning of metamorphosis. Although specific patterns of PCD were different in distantly related ascidian species, the results suggest that removal of CNS cells by apoptosis is a urchordate feature predating the origin of the vertebrates. Certain molgulid ascidian species have evolved an anural (tailless) larva in which notochord cells fail to undergo the morphogenetic movements culminating in tail development. These anural species include Molgula occulta, the sister species of the urodele (tailed) species Molgula oculata. We show that PCD in the notochord cell lineage precedes the arrest of tail development in M. occulta and other independently evolved anural species. The notochord cells are rescued from PCD and a tail develops in hybrid embryos produced by fertilizing M. occulta eggs with M. oculata sperm, implying that apoptosis is controlled zygotically. Antisense inhibition experiments show that zygotic expression of the FoxA5 and Manx genes is required to prevent notochord PCD in urodele species and hybrids with restored tails. The results provide the first indication of PCD in the ascidian embryo and suggest that apoptosis modulated by FoxA5 and Manx is involved in notochord and tail regression during anural development. Differences in PCD that occur between ascidian species suggest that diversity in programming apoptosis may explain differences in larval form.     

Autophagy and apoptosis coordinate physiological cell death in larval salivary glands of Apis mellifera (Hymenoptera: Apidae)

Larval salivary glands of bees provide a good model for the study of hormone-induced programmed cell death in Hymenoptera because they have a well-defined secretory cycle with a peak of secretory activity phase, prior to cocoon spinning, and a degenerative phase, after the cocoon spinning. Our findings demonstrate that there is a relationship between apoptosis and autophagy during physiological cell death in these larval salivary glands, that adds evidence to the hypothesis of overlap in the regulation pathways of both types of programmed cell death. Features of autophagy include cytoplasm vacuolation, acid phosphatase activity, presence of autophagic vacuoles and multi-lamellar structures, as well as a delay in the collapse of many nuclei. Features of apoptosis include bleb formation in the cytoplasm and nuclei, with release of parts of the cytoplasm into the lumen, chromatin compaction, and DNA and nucleolar fragmentation. We propose a model for programmed cell death in larval salivary glands of Apis mellifera where autophagy and apoptosis function cooperatively for a more efficient degeneration of the gland secretory cells.     

Developmental Studies in Drosophila

The salivary gland chromosomes of 3rd instar Drosophila pseudoobscura larvae were observed for puffing changes after injection of larvae with ecdysterone solution. Chromosomes from the salivary glands of 3rd instar larvae and prepupae were similarly examined after incubation in ecdysterone-containing medium. The larvae, after treatment, showed advancement of the puffing process with the occurrence of a pattern similar to that observed during the pre-spiracle eversion period of normal development. At least 92 puffs showed changes in size. For the prepupae, the puffing changes resembled those occurring normally during the late prepupal period. A group of puffs were selected for detailed study. Among these were four puffs on the XR chromosome which exhibited large increases before spiracle eversion and pupation in normal development. As in normal development, two of these became the most prominent puffs observed within h after hormone treatment. In chromosomes from larval glands, the other two XR chromosome puffs were among the largest puffs to appear later in the sequence. However, in chromosomes from prepupal glands one of these later puffs failed to appear. The significance of this large number of hormone-inducible puffing changes at two different periods in development is discussed.     

果蝇变态过程中的细胞凋亡和细胞自噬

程序化细胞死亡(programmed cell death,PCD)分为I型PCD细胞凋亡(apoptosis)和II型PCD细胞自噬(autophagy)。果蝇等完全变态昆虫有2种类型的器官:即细胞内分裂器官(如脂肪体、表皮、唾液腺、中肠、马氏管等)和有丝分裂器官(复眼、翅膀、足、神经系统等)。在昆虫变态过程中,细胞内分裂器官进行器官重建,幼虫器官大量发生细胞凋亡和细胞自噬到最后完全消亡,同时成虫器官由干细胞从新生成;而有丝分裂器官则由幼虫器官直接发育为成虫器官。在果蝇等昆虫的变态过程中,细胞凋亡和细胞自噬在幼虫器官的死亡和成虫器官的生成中发挥了非常重要的作用。文章简要介绍细胞凋亡和细胞自噬在果蝇变态过程中的生理功能和分子调控机制。     

Many ways to exit? Cell death categories in plants

Programmed cell death (PCD) is an integral part of plant development and defence. It occurs at all stages of the life cycle, from fertilization of the ovule to death of the whole plant. Without it, tall trees would probably not be possible and plants would more easily succumb to invading microorganisms. Here, we have attempted to categorize plant PCD in relation to three established morphological types of metazoan cell death: apoptosis, autophagy and non-lysosomal PCD. We conclude that (i) no examples of plant PCD conform to the apoptotic type, (ii) many examples of PCD during plant development agree with the autophagic type, and (iii) that other examples are apparently neither apoptotic nor autophagic.     

The Drosophila melanogaster Apaf-1 homologue ARK is required for most, but not all, programmed cell death

The Apaf-1 protein is essential for cytochrome c-mediated caspase-9 activation in the intrinsic mammalian pathway of apoptosis. Although Apaf-1 is the only known mammalian homologue of the Caenorhabditis elegans CED-4 protein, the deficiency of apaf-1 in cells or in mice results in a limited cell survival phenotype, suggesting that alternative mechanisms of caspase activation and apoptosis exist in mammals. In Drosophila melanogaster, the only Apaf-1/CED-4 homologue, ARK, is required for the activation of the caspase-9/CED-3-like caspase DRONC. Using specific mutants that are deficient for ark function, we demonstrate that ARK is essential for most programmed cell death (PCD) during D. melanogaster development, as well as for radiation-induced apoptosis. ark mutant embryos have extra cells, and tissues such as brain lobes and wing discs are enlarged. These tissues from ark mutant larvae lack detectable PCD. During metamorphosis, larval salivary gland removal was severely delayed in ark mutants. However, PCD occurred normally in the larval midgut, suggesting that ARK-independent cell death pathways also exist in D. melanogaster.     

Identification of a Drosophila melanogaster ICE/CED-3-related protease, drICE.

Cysteine proteases of the ICE/CED-3 family (caspases) are required for the execution of programmed cell death (PCD) in a wide range of multicellular organisms. Caspases are implicated in the execution of apoptosis in Drosophila melanogaster by the observation that expression of baculovirus p35, a caspase inhibitor, blocks cell death in vivo in Drosophila. We report here the identification and characterization of drICE, a D. melanogaster caspase. We show that overexpression of drICE sensitizes Drosophila cells to apoptotic stimuli and that expression of an N-terminally truncated form of drICE rapidly induces apoptosis in Drosophila cells. Induction of apoptosis by rpr overexpression or by cycloheximide or etoposide treatment of Drosophila cells results in proteolytic processing of drICE. We further show that drICE is a cysteine protease that cleaves baculovirus p35 and Drosophila lamin DmO in vitro and that drICE is expressed at all the stages of Drosophila development at which PCD can be induced. Taken together, these results strongly argue that drICE is an apoptotic caspase that acts downstream of rpr. drICE is therefore the first unequivocal link between the molecular machinery of Drosophila cell death and the conserved machinery of Caenorhabditis elegans and vertebrates. Identification of drICE should facilitate the elucidation of upstream regulators and downstream targets of caspases by genetic screening.     

Rearrangement of the tubulin and actin cytoskeleton during programmed cell death in Drosophila salivary glands

During larva-to-pupa metamorphosis Drosophila salivary glands undergo programmed cell death by autophagocytosis. Although ultrastructure of Drosophila salivary glands has been extensively studied in the past, little is known about mechanism of programmed cell death, especially the role of the cytoskeleton. In this paper we describe changes in microtubule and actin filament network compared to the progress of DNA fragmentation and redistribution of acid phosphatase. In feeding and wandering larvae microtubules and actin filaments form regular networks localized mostly along the plasma membrane. The first major rearrangement of microtubules and actin filaments occurred when larvae everted spiracles and the glands shifted their secretion from saliva to mucoprotein glue (stage L1). Microtubule cytoskeleton became denser and actin filaments concentrated along cell boundaries. At the same time nuclei flattened and migrated into the microtubule-rich layer near the basal membrane. In late prepupae (8-10 h after P1) the microtubule network became fainter, and actin filaments appeared frequently deeper in cytoplasm, gradually concentrating around nuclei. Simultaneously large patches of acid phosphatase activity surrounded nuclei and shortly thereafter chromosomal DNA began to fragment. During the final collapse of the gland (early pupae, 13.5 h after formation of white puparium) cellular fragments and autophagic vacuoles contained a continuous F-actin lining and the microtubule network displayed signs of extensive degradation. The results are consistent with the hypothesis that, in Drosophila salivary glands, extensive autophagic activities target nuclei for degradation; that this process occurs late in the course of programmed cell death; and that it directly involves cytoskeletal structures which are altered far earlier during the course of cell death.     

Mechanisms of programmed cell death in the midgut and salivary glands from Bradysia hygida (Diptera: Sciaridae) during pupal–adult metamorphosis

Programmed cell death is involved with the degeneration/remodeling of larval tissues and organs during holometabolous development. The midgut is a model to study the types of programmed cell death associated with metamorphosis because its structure while degenerating is a substrate for the formation of the adult organ. Another model is the salivary glands from dipteran because their elimination involves different cell death modes. This study aimed to investigate the models of programmed cell death operating during midgut replacement and salivary gland histolysis in Bradysia hygida. We carried out experiments of real‐time observations, morphological analysis, glycogen detection, filamentous‐actin localization, and nuclear acridine orange staining. Our findings allow us to establish that an intact actin cytoskeleton is required for midgut replacement in B. hygida and nuclear condensation and acridine orange staining precede the death of the larval cells. Salivary glands in histolysis present cytoplasmic blebbing, nuclear retraction, and acridine orange staining. This process can be partially reproduced in vitro. We propose that the larval midgut death involves autophagic and apoptotic features and apoptosis is a mechanism involved with salivary gland histolysis.