Molecular mechanisms of cell death and phagocytosis in Drosophila

The genetic tools available in Drosophila have facilitated our understanding of how apoptosis is regulated and executed in the context of the developing organism. All embryonic apoptosis is initiated by the activity of three genes, rpr, grim and hid. Each of these genes is independently regulated, allowing developmental apoptosis to be finely controlled. These initiators in turn activate the core apoptotic machinery, including the caspases. Drosophila counterparts to other conserved components of the apoptotic machinery have been recently identified, and we discuss how these may be integrated into the process of normal developmentally regulated cell death. We also outline the role that phagocytosis plays in the final stages of apoptosis and consider the molecular mechanisms guiding the elimination of apoptotic corpses.     

Coordinated expression of cell death genes regulates neuroblast apoptosis

Properly regulated apoptosis in the developing central nervous system is crucial for normal morphogenesis and homeostasis. In Drosophila, a subset of neural stem cells, or neuroblasts, undergo apoptosis during embryogenesis. Of the 30 neuroblasts initially present in each abdominal hemisegment of the embryonic ventral nerve cord, only three survive into larval life, and these undergo apoptosis in the larvae. Here, we use loss-of-function analysis to demonstrate that neuroblast apoptosis during embryogenesis requires the coordinated expression of the cell death genes grim and reaper, and possibly sickle. These genes are clustered in a 140 kb region of the third chromosome and show overlapping patterns of expression. We show that expression of grim, reaper and sickle in embryonic neuroblasts is controlled by a common regulatory region located between reaper and grim. In the absence of grim and reaper, many neuroblasts survive the embryonic period of cell death and the ventral nerve cord becomes massively hypertrophic. Deletion of grim alone blocks the death of neuroblasts in the larvae. The overlapping activity of these multiple cell death genes suggests that the coordinated regulation of their expression provides flexibility in this crucial developmental process.     

Overlapping functions of microRNAs in control of apoptosis during Drosophila embryogenesis

Regulation of apoptosis is crucial for tissue homeostasis under normal development and environmental stress. In Drosophila, cell death occurs in different developmental processes including embryogenesis. Here, we report that two members of the miR-2 seed family of microRNAs, miR-6 and miR-11, function together to limit the level of apoptosis during Drosophila embryonic development. Mutants lacking both miR-6 and miR-11 show embryonic lethality and defects in the central nervous system (CNS). We provide evidence that miR-6/11 functions through regulation of the proapoptotic genes, reaper (rpr), head involution defective (hid), grim and sickle (skl). Upregulation of these proapoptotic genes is responsible for the elevated apoptosis and the CNS defects in the mutants. These findings demonstrate that the activity of the proapoptotic genes is kept in check by miR-6/11 to ensure normal development.     

Commitment of abdominal neuroblasts in Drosophila to a male or female fate is dependent on genes of the sex-determining hierarchy.

Adult specific neurons in the central nervous system of holometabolous insects are generated by the postembryonic divisions of neuronal stem cells (neuroblasts). In the ventral nervous system of Drosophila melanogaster, sex-specific divisions by a set of abdominal neuroblasts occur during larval and early pupal stages. Animals mutant for several sex-determining genes were analyzed to determine the genetic regulation of neuroblast commitment to the male or female pattern of division and the time during development when these decisions are made. We have found that the choice of the sexual pathway taken by sex-specific neuroblasts depends on the expression of one of these genes, doublesex (dsx). In the absence of any functional dxs+ products, the sex-specific neuroblasts fail to undergo any postembryonic divisions in male or female larval nervous systems. From the analysis of intersexes generated by dominant alleles of dsx, it has been concluded that the same neuroblasts provide the sex-specific neuroblasts in both male and female central nervous systems. The time when neuroblasts become committed to generate their sex-specific divisions were identified by shifting tra-2ts flies between the male- and female-specifying temperatures at various times during larval development. Neuroblasts become determined to adopt a male or female state at the end of the first larval instar, a time when abdominal neuroblasts enter their first postembryonic S-phase.     

Expression and function of the Drosophila gene runt in early stages of neural development.

The Drosophila gene runt was initially identified on the basis of its role during segmentation. Recent molecular and genetic studies have demonstrated that the runt gene encodes a novel nuclear protein whose developmental importance is not exclusive to segmentation. This report addresses the functional relevance of runt expression in the developmental pathway of neurogenesis. Antibodies against the runt protein reveal that it is expressed in a subset of neuroblasts, ganglion-mother cells and neurons. A subset of these neurons also co-express the segmentation gene even-skipped (eve). Using eve as a marker, we show that runt is required for the normal development of these neurons. A runt P-transposon that lacks neural cis-regulatory elements is used to show that these neurons require runt activity independent of its activity during segmentation. These results are confirmed using a temperature-sensitive runt allele. Further temperature-shift experiments indicate that the requirement for runt is during an early stage of neurogenesis. Based on its pattern of expression and its temporal requirements, runt is distinguished as one of the earliest acting genes involved in the generation of diverse cell fates in the developing Drosophila nervous system.     

The RET proto-oncogene induces apoptosis: a novel mechanism for Hirschsprung disease

The RET (rearranged during transfection) proto-oncogene encodes a tyrosine kinase receptor involved in both multiple endocrine neoplasia type 2 (MEN 2), an inherited cancer syndrome, and Hirschsprung disease (HSCR), a developmental defect of enteric neurons. We report here that the expression of RET receptor induces apoptosis. This pro-apoptotic effect of RET is inhibited in the presence of its ligand glial cell line-derived neurotrophic factor (GDNF). Furthermore, we present evidence that RET induces apoptosis via its own cleavage by caspases, a phenomenon allowing the liberation/exposure of a pro-apoptotic domain of RET. In addition, we report that Hirschsprung-associated RET mutations impair GDNF control of RET pro-apoptotic activity. These results indicate that HSCR may result from apoptosis of RET-expressing enteric neuroblasts.     

Debcl, a proapoptotic Bcl-2 homologue, is a component of the Drosophila melanogaster cell death machinery

Bcl-2 family of proteins are key regulators of apoptosis. Both proapoptotic and antiapoptotic members of this family are found in mammalian cells, but no such proteins have been described in insects. Here, we report the identification and characterization of Debcl, the first Bcl-2 homologue in Drosophila melanogaster. Structurally, Debcl is similar to Bax-like proapoptotic Bcl-2 family members. Ectopic expression of Debcl in cultured cells and in transgenic flies causes apoptosis, which is inhibited by coexpression of the baculovirus caspase inhibitor P35, indicating that Debcl is a proapoptotic protein that functions in a caspase-dependent manner. debcl expression correlates with developmental cell death in specific Drosophila tissues. We also show that debcl genetically interacts with diap1 and dark, and that debcl-mediated apoptosis is not affected by gene dosage of rpr, hid, and grim. Biochemically, Debcl can interact with several mammalian and viral prosurvival Bcl-2 family members, but not with the proapoptotic members, suggesting that it may regulate apoptosis by antagonizing prosurvival Bcl-2 proteins. RNA interference studies indicate that Debcl is required for developmental apoptosis in Drosophila embryos. These results suggest that the main components of the mammalian apoptosis machinery are conserved in insects.     

Biochemical and genetic interactions between Drosophila caspases and the proapoptotic genes rpr, hid, and grim

In Drosophila melanogaster, the induction of apoptosis requires three closely linked genes, reaper (rpr), head involution defective (hid), and grim. The products of these genes induce apoptosis by activating a caspase pathway. Two very similar Drosophila caspases, DCP-1 and drICE, have been previously identified. We now show that DCP-1 has a substrate specificity that is remarkably similar to those of human caspase 3 and Caenorhabditis elegans CED-3, suggesting that DCP-1 is a death effector caspase. drICE and DCP-1 have similar yet different enzymatic specificities. Although expression of either in cultured cells induces apoptosis, neither protein was able to induce DNA fragmentation in Drosophila SL2 cells. Ectopic expression of a truncated form of dcp-1 (DeltaN-dcp-1) in the developing Drosophila retina under an eye-specific promoter resulted in a small and rough eye phenotype, whereas expression of the full-length dcp-1 (fl-dcp-1) had little effect. On the other hand, expression of either full-length drICE (fl-drICE) or truncated drICE (DeltaN-drICE) in the retina showed no obvious eye phenotype. Although active DCP-1 protein cleaves full-length DCP-1 and full-length drICE in vitro, GMR-DeltaN-dcp-1 did not enhance the eye phenotype of GMR-fl-dcp-1 or GMR-fl-drICE flies. Significantly, GMR-rpr and GMR-grim, but not GMR-hid, dramatically enhanced the eye phenotype of GMR-fl-dcp-1 flies. These results indicate that Reaper and Grim, but not HID, can activate DCP-1 in vivo.     

Neuroblast mitosis in dissociated culture: regulation and relationship to differentiation

Although neuron generation is precisely regulated during ontogeny, little is known about underlying mechanisms. In addition, relationships between precursor proliferation and the apparent sequence of developmental processes, including cell migration, neurite elaboration, transmitter expression and synaptogenesis remain unknown. To address these issues, we used a fully defined neuronal cell culture system derived from embryonic rat sympathetic ganglia (DiCicco-Bloom, E., and I. B. Black. 1988. Proc. Natl. Acad. Sci. USA. 85:4066-4070) in which precursors enter the mitotic cycle. We now find that, in addition to synthesizing DNA, neuroblasts also underwent division in culture, allowing analysis of developmental relationships and mitotic regulation. Our observations indicate that mitotic neuroblasts expressed a wide array of neuron-specific characteristics including extension of neuritic processes with growth cones, elaboration of neurotransmitter enzyme, synthesis and transport of transmitter vesicles and organization of transmitter release sites. These data suggest that neuroblasts in the cell cycle may simultaneously differentiate. Consequently, the apparent sequence of ontogenetic processes is not an immutable, intrinsic neuronal program. How, then, are diverse developmental events coordinated? Our observations indicate that neuroblast mitosis is regulated by a small number of epigenetic factors, including insulin and EGF. Since these signals also influence other processes in developing neurons, epigenetic regulation normally may synchronize diverse ontogenetic events.     

Drosophila Bcl-2 proteins participate in stress-induced apoptosis, but are not required for normal development

Many developing tissues require programmed cell death (PCD) for proper formation. In mice and C. elegans, developmental PCD is regulated by the Bcl-2 family of proteins. Two bcl-2 genes are encoded in the Drosophila genome (debcl/dBorg1/Drob-1/dBok and buffy/dBorg2) and previous RNAi-based studies suggested a requirement for these in embryonic development. However, we report here that, despite the fact that many tissues in fruit flies are shaped by PCD, deletion of the bcl-2 genes does not perturb normal development. We investigated whether the fly bcl-2 genes regulate non-apoptotic processes that require caspases, but found these to be bcl-2 gene-independent. However, irradiation of the mutants demonstrates that DNA damage-induced apoptosis, mediated by Reaper, is blocked by buffy and that debcl is required to inhibit buffy. Our results demonstrate that developmental PCD regulation in the fly does not rely upon the Bcl-2 proteins, but that they provide an added layer of protection in the apoptotic response to stress.     

Progenitor properties of symmetrically dividing Drosophila neuroblasts during embryonic and larval development

Asymmetric cell division generates two daughter cells of differential gene expression and/or cell shape. Drosophila neuroblasts undergo typical asymmetric divisions with regard to both features; this is achieved by asymmetric segregation of cell fate determinants (such as Prospero) and also by asymmetric spindle formation. The loss of genes involved in these individual asymmetric processes has revealed the roles of each asymmetric feature in neurogenesis, yet little is known about the fate of the neuroblast progeny when asymmetric processes are blocked and the cells divide symmetrically. We genetically created such neuroblasts, and found that in embryos, they were initially mitotic and then gradually differentiated into neurons, frequently forming a clone of cells homogeneous in temporal identity. By contrast, larval neuroblasts with the same genotype continued to proliferate without differentiation. Our results indicate that asymmetric divisions govern lineage length and progeny fate, consequently generating neural diversity, while the progeny fate of symmetrically dividing neuroblasts depends on developmental stages, presumably reflecting differential activities of Prospero in the nucleus.     

Restriction of developmental potential during divergence of the enteric and sympathetic neuronal lineages.

In the peripheral nervous system, enteric and sympathetic neurons develop from multipotent neural crest cells. While local environmental signals in the gut and in the region of the sympathetic ganglia play a role in the choice of cell fate, little is known about the mechanisms that underlie restriction to specific neuronal phenotypes. We investigated the divergence and restriction of the enteric and sympathetic neuronal lineages using immuno-isolated neural crest-derived cells from the gut and sympathetic ganglia. Analysis of neuronal and lineage-specific mRNAs and proteins indicated that neural crest-derived cells from the gut and sympathetic ganglia had initiated neuronal differentiation and phenotypic divergence by E14.5 in the rat. We investigated the developmental potential of these cells using expression of tyrosine hydroxylase as a marker for a sympathetic phenotype. Tyrosine hydroxylase expression was examined in neurons that developed from sympathetic and enteric neuroblasts under the following culture conditions: culture alone; coculture with gut monolayers to promote enteric differentiation; or coculture with dorsal aorta monolayers to promote noradrenergic differentiation. Both enteric and sympathetic neuroblasts displayed developmental plasticity at E14.5. Sympathetic neuroblasts downregulated tyrosine hydroxylase in response to signals from the gut environment and enteric neuroblasts increased expression of tyrosine hydroxylase when grown on dorsal aorta or in the absence of other cell types. Tracking of individual sympathetic cells displaying a neuronal morphology at the time of plating indicated that neuroblasts retained phenotypic plasticity even after initial neuronal differentiation had occurred. By E19.5 both enteric and sympathetic neuroblasts had undergone a significant loss of their developmental potential, with most neuroblasts retaining their lineage-specific phenotype in all environments tested. Together our data indicate that the developmental potential of enteric and sympathetic neuroblasts becomes restricted over time and that this restriction takes place not as a consequence of initial neuronal differentiation but during the period of neuronal maturation. Further, we have characterized a default pathway of adrenergic differentiation in the enteric nervous system and have defined a transient requirement for gut-derived factors in the maintenance of the enteric neuronal phenotype.     

ATM is required for telomere maintenance and chromosome stability during Drosophila development

ATM is a large, multifunctional protein kinase that regulates responses required for surviving DNA damage: including DNA repair, apoptosis, and cell cycle checkpoints. Here, we show that Drosophila ATM function is essential for normal adult development. Extensive, inappropriate apoptosis occurs in proliferating atm mutant tissues, and in clonally derived atm mutant embryos, frequent mitotic defects were seen. At a cellular level, spontaneous telomere fusions and other chromosomal abnormalities are common in atm larval neuroblasts, suggesting a conserved and essential role for dATM in the maintenance of normal telomeres and chromosome stability. Evidence from other systems supports the idea that DNA double-strand break (DSB) repair functions of ATM kinases promote telomere maintenance by inhibition of illegitimate recombination or fusion events between the legitimate ends of chromosomes and spontaneous DSBs. Drosophila will be an excellent model system for investigating how these ATM-dependent chromosome structural maintenance functions are deployed during development. Because neurons appear to be particularly sensitive to loss of ATM in both flies and humans, this system should be particularly useful for identifying cell-specific factors that influence sensitivity to loss of dATM and are relevant for understanding the human disease, ataxia-telangiectasia.     

Aging rate after continual low dose irradiation of drosophila strains with apoptosis deregulation

In this article the results of investigations of interrelation between radio-induced apoptosis in larvae nervous system and aging in imago are presented. It was shown that the apoptosis level of larvae nervous ganglion 2.5 folds increased after exposure to ionizing radiation (54 cGy). Irradiation of stocks with higher apoptosis induction sensitivity, bearing mutations of inhibitor apoptosis protein th (allele th1 and th4) and wild type strain Berlin leaded to elevation of activity at all ages and accordingly to decreasing of aging speed. Whereas in stocks with lower apoptosis induction sensitivity (with mutations of genes rpr, grim, hid, dArk and Dcp-1) this effect was not observed. The obtained results suggested that radiation-induced apoptosis could be one of the mechanisms preventing aging.