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Methods for studying apoptosis and phagocytosis of apoptotic cells in Drosophila tissues and cell lines
... Therefore, we did TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay to test if newt genes can block or downregulate cell death to rescue L 2 mutant phenotype (Figures 3G-3L and S5J-S5R). TUNEL assay is based on the detection of fragmented DNA ends, which are characteristic of apoptotic cells (Sarkissian et al., 2014;White et al., 2001). The number of dying cells are compared between the L 2 mutant, and the L 2 mutant background where the newt gene was misexpressed. ...
... TUNEL assay for detection of cell death Dissection and immunostaining before TUNEL labeling. Apoptosis was detected by using Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay White et al., 2001). TUNEL assay is based on the detection of fragmented DNA ends, which are characteristic of apoptotic cells. ...
Newts utilize their unique genes to restore missing parts by strategic regulation of conserved signaling pathways. Lack of genetic tools pose challenges to determine the function of such genes. Therefore, we used the Drosophila eye model to demonstrate the potential of 5 unique newt (Notophthalmus viridescens) gene(s), viropana1-viropana5 (vna1-vna5), which were ectopically expressed in L² mutant and GMR-hid, GMR-GAL4 eye. L² exhibits the loss of ventral half of early eye and head involution defective (hid) triggers cell-death during later eye development. Surprisingly newt genes significantly restore missing photoreceptor cells both in L² and GMR>hid background by upregulating cell-proliferation and blocking cell-death, regulating evolutionarily conserved Wingless (Wg)/Wnt signaling pathway and exhibit non-cell-autonomous rescues. Further, Wg/Wnt signaling acts downstream of newt genes. Our data highlights that unique newt proteins can regulate conserved pathways to trigger a robust restoration of missing photoreceptor cells in Drosophila eye model with weak restoration capability.
... Ectopic buffy can inhibit cell cycle progression (Quinn et al., 2003); however, no compensatory increase in cell division was observed in the buffy mutant ( Supplementary Fig 2). To directly assess death due to Grim expression in the eye, we assayed retention of Acridine Orange, a commonly used marker of dying cells in Drosophila (White et al., 2001). The increase in cell death seen by expression of grim is reduced in the absence of buffy ( Fig 4D, compare panels 2 and 3). ...
The Inhibitor of apoptosis (IAP) antagonists Reaper (Rpr), Grim and Hid are central regulators of developmental apoptosis in Drosophila. Ectopic expression of each is sufficient to trigger apoptosis, and hid and rpr have been shown to be important for programmed cell death (PCD). To investigate the role for grim in PCD, a grim null mutant was generated. grim was not a key proapoptotic gene for embryonic PCD, confirming that grim cooperates with rpr and hid in embryogenesis. In contrast, PCD of glial cells in the microchaete lineage required grim, identifying a death process dependent upon endogenous grim. Grim associates with mitochondria and has been shown to activate a mitochondrial death pathway distinct from IAP antagonization; therefore, the Drosophila bcl-2 genes buffy and debcl were investigated for genetic interaction with grim. Loss of buffy led to microchaete glial cell survival and suppressed death in the eye induced by ectopic Grim. This is the first example of a developmental PCD process influenced by buffy, and places buffy in a proapoptotic role. PCD of microchaete glial cells represents an exceptional opportunity to study the mitochondrial proapoptotic process induced by Grim.
... Discs were mounted in Vectashield (Vector Labs). Apoptosis was detected using Apoptag Red (Intergen) reagents as described (White et al., 2001). ...
It is largely unknown how growth slows and then stops in vivo. Similar to most organs, Drosophila imaginal discs undergo a fast, near-exponential growth phase followed by a slow growth phase before final target size is reached. We have used a genetic approach to study the role of an ABC-E protein, Pixie, in wing disc growth. pixie mutants, like mutants in ribosomal proteins genes (known as Minutes), show severe developmental delay with relatively mild alterations in final body size. Intriguingly, pixie mutant wing imaginal discs show complex regional and temporal defects in growth and cell survival that are compensated to result in near-normal final size. In S2 cells, Pixie, like its yeast homolog RLI1, is required for translation. However, a comparison of the growth of eukaryotic translation initiation factor eIF4A and pixie mutant clones in wing discs suggests that only a subset of translation regulators, including pixie, mediate regional differences in growth and cell survival in wing discs. Interestingly, some of the regional effects on pixie mutant clone growth are enhanced in a Minute background. Our results suggest that the role of Pixie is not merely to allow growth, as might be expected for a translation regulator. Instead, Pixie also behaves as a target of putative constraining signals that slow disc growth during late larval life. We propose a model in which a balance of growth inhibitors and promoters determines tissue growth rates and cell survival. An alteration in this balance slows growth before final disc size is reached.
... Embryos were collected and aged until reaching stage 13 (8.5-10.5 hr after egg laying). Acridine orange (AO) staining was performed as previously described (White et al., 2001). ...
Many cells die by apoptosis during animal development. Apoptotic cells are rapidly removed through phagocytosis by their neighbors or by macrophages. To genetically dissect this process, we performed an in vivo screen for genes required for efficient phagocytosis of apoptotic cells by Drosophila macrophages and identified pallbearer (pall), which encodes an F box protein. F box proteins generally provide substrate specificity to Skp Cullin F box (SCF) complexes, acting as E3 ligases that target phosphorylated proteins to ubiquitylation and degradation via the 26S proteasome. We showed that Pallbearer functions in an SCF-dependent manner and provided direct evidence of a role for ubiquitylation and proteasomal degradation in phagocytosis of apoptotic corpses in vivo. This work might further our understanding of the regulation of apoptotic cell engulfment and thus our understanding of innate immunity as a whole.
The Drosophila wing somatic mutation and recombination test (SMART; also known as the wing spot test) provides a rapid means to assess the potential of a chemical to induce loss of heterozygosity (LOH) resulting from gene mutation, chromosomal rearrangement, chromosome breakage, or chromosome loss. This bioassay makes use of the wing-cell recessive markers multiple wing hairs mwh, 3–0.3) and flare (flr 3, 3–38.8) in transheterozygous mwh +/+ flr 3 animals. When a genetic alteration is induced in a mitotically dividing cell of a developing wing disc, it may give rise to a clone(s) of mwh and/or flr 3 cells (i.e., a “spot”) visible on the wing surface of the adult fly. The total number of clones induced in a group of chemically treated flies gives quantitative data concerning the whole genotoxic activity of a compound, whereas the types of clone can reveal the mutational mechanisms involved in clone production. Variations on the SMART system described in this chapter have been used to measure frequencies of spontaneous LOH in mutants defective in meiotic recombination and disjunction, DNA repair, and cell proliferation (1–3).
Billions of cells die and are cleared throughout the development and homeostasis of an organism. Either improper death or clearance can lead to serious illnesses. In the adult Drosophila ovary, germline cells can die by programmed cell death (PCD) at three distinct stages; here we focus on cell death that occurs in mid- and late oogenesis. In mid-oogenesis, the germline of egg chambers can undergo apoptosis in response to nutrient deprivation. In late oogenesis, the nurse cells are eliminated through a developmentally regulated, non-apoptotic cell death. In this chapter, we describe several methods to detect cell death and phagocytosis in the Drosophila ovary. DAPI stains the chromatin of all cells and can be used to detect morphological changes in cells that die by different mechanisms. TUNEL labels fragmented DNA, which can occur in both apoptotic and non-apoptotic death. LysoTracker, an acidophilic dye, marks acidic vesicles and some dying cells; therefore, it can be used to study both death and phagocytosis. We also describe several antibodies that can be used to investigate cell death and/or phagocytosis: active caspase Dcp-1, membrane markers, and lamins. Many of these antibodies can be used in combination with GFP fusion transgenes for further analysis; we show Rab5-GFP and Rab7-GFP, which can be used to study phagocytosis in further detail.
The fruit fly, Drosophila melanogaster, has been instrumental in identifying pathways regulating development and signal transduction. Drosophila has been a relatively late entrant into the world of apoptosis, but with its powerful genetic tools, studies in the fruit
fly are destined to contribute essential insights into cell death. Drosophila has homologues to mammalian apoptotic genes, and their function can be studied at the cellular level as well as in the developing
organism. As in other organisms, cell death in the fruit fly removes damaged and unneeded cells. Beyond this, however, experiments
in the fruit fly have provided unique insights into how cell death removes cells that initiate the incorrect developmental
program. Furthermore, Drosophila studies have begun to elucidate how cell death is spatially regulated for precise pattern formation. In this chapter, we
introduce the molecular players in Drosophila cell death, discuss current knowledge about cell death during development, introduce the idea that dying cells induce neighboring
cells to proliferate, and finish with nonapoptotic roles for cell death proteins.
Programmed cell death (PCD) is an essential and wide-spread physiological process that results in the elimination of cells. Genes required to carry out this process have been identified, and many of these remain the subjects of intense investigation. Here, we describe PCD, its functions, and some of the consequences when it goes awry. We review PCD in the model system, the fruit fly, Drosophila melanogaster, with a particular emphasis on cell death gene discovery resulting from both genetics and genomics-based approaches.
Mutagenesis screens in zebrafish have uncovered several hundred mutant alleles affecting the development of the retina and established the zebrafish as one of the leading models of vertebrate eye development. In addition to forward genetic mutagenesis approaches, gene function in the zebrafish embryo is being studied using several reverse genetic techniques. Some of these rely on the overexpression of a gene product, others take advantage of antisense oligonucleotides to block function of selected loci. Here we describe these methods in the context of the developing eye.
The JAK/STAT signalling pathway mediates both antiproliferative responses following interferon stimulation and cellular proliferation in response to cytokines such as interleukins and growth factors. Central to these responses are the seven vertebrate STAT molecules, misregulation of which is implicated in a variety of malignancies. We have investigated the proliferative role of the single Drosophila STAT92E, part of the evolutionarily conserved JAK/STAT cascade. During second instar larval wing disc development pathway activity is both necessary and sufficient to promote proliferation of this epithelial cell type. However by later stages, endogenous STAT92E is stimulated by a noncannonical mechanism to exert pronounced antiproliferative effects. Ectopic canonical activation is sufficient to further decrease proliferation and leads to the premature arrest of cells in the G2 phase of the cell cycle. The single STAT92E present in Drosophila therefore mediates both proproliferative functions analogous to vertebrate interleukin-stimulated STAT3 and antiproliferative functions analogous to interferon-stimulated STAT1. Pro- and antiproliferative roles therefore represent ancestral activities conserved through evolution and subsequently assigned to distinct molecules.
Programmed cell death (PCD) plays a key role in developmental biology and in maintenance of the steady state in continuously renewing tissues. Currently, its existence is inferred mainly from gel electrophoresis of a pooled DNA extract as PCD was shown to be associated with DNA fragmentation. Based on this observation, we describe here the development of a method for the in situ visualization of PCD at the single-cell level, while preserving tissue architecture. Conventional histological sections, pretreated with protease, were nick end labeled with biotinylated poly dU, introduced by terminal deoxy-transferase, and then stained using avidin-conjugated peroxidase. The reaction is specific, only nuclei located at positions where PCD is expected are stained. The initial screening includes: small and large intestine, epidermis, lymphoid tissues, ovary, and other organs. A detailed analysis revealed that the process is initiated at the nuclear periphery, it is relatively short (1-3 h from initiation to cell elimination) and that PCD appears in tissues in clusters. The extent of tissue-PCD revealed by this method is considerably greater than apoptosis detected by nuclear morphology, and thus opens the way for a variety of studies.
Peroxidasin is a novel protein combining peroxidase and extracellular matrix motifs. Hemocytes differentiate early from head mesoderm, make peroxidasin and later phagocytose apoptotic cells. As hemocytes spread throughout the embryo, they synthesize extracellular matrix and peroxidasin, incorporating it into completed basement membranes. Cultured cells secrete peroxidasin; it occurs in larvae and adults. Each 1512 residue chain of the three-armed, disulfide-linked homotrimer combines a peroxidase domain with six leucine-rich regions, four Ig loops, a thrombospondin/procollagen homology and an amphipathic alpha-helix. The peroxidase domain is homologous with human myeloperoxidase and eosinophil peroxidase. This heme protein catalyzes H2O2-driven radioiodinations, oxidations and formation of dityrosine. We propose that peroxidasin functions uniquely in extracellular matrix consolidation, phagocytosis and defense.
A gene, reaper (rpr), that appears to play a central control function for the initiation of programmed cell death (apoptosis)
in Drosophila was identified. Virtually all programmed cell death that normally occurs during Drosophila embryogenesis was
blocked in embryos homozygous for a small deletion that includes the reaper gene. Mutant embryos contained many extra cells
and failed to hatch, but many other aspects of development appeared quite normal. Deletions that include reaper also protected
embryos from apoptosis caused by x-irradiation and developmental defects. However, high doses of x-rays induced some apoptosis
in mutant embryos, and the resulting corpses were phagocytosed by macrophages. These data suggest that the basic cell death
program is intact although it was not activated in mutant embryos. The DNA encompassed by the deletion was cloned and the
reaper gene was identified on the basis of the ability of cloned DNA to restore apoptosis to cell death defective embryos
in germ line transformation experiments. The reaper gene appears to encode a small peptide that shows no homology to known
proteins, and reaper messenger RNA is expressed in cells destined to undergo apoptosis.
The deliberate and orderly removal of cells by programmed cell death is a common phenomenon during the development of metazoan animals. We have examined the distribution and ultrastructural appearance of cell deaths that occur during embryogenesis in Drosophila melanogaster. A large number of cells die during embryonic development in Drosophila. These cells display ultrastructural features that resemble apoptosis observed in vertebrate systems, including nuclear condensation, fragmentation and engulfment by macrophages. Programmed cell deaths can be rapidly and reliably visualized in living wild-type and mutant Drosophila embryos using the vital dyes acridine orange or nile blue. Acridine orange appears to selectively stain apoptotic forms of death in these preparations, since cells undergoing necrotic deaths were not significantly labelled. Likewise, toluidine blue staining of fixed tissues resulted in highly specific labelling of apoptotic cells, indicating that apoptosis leads to specific biochemical changes responsible for the selective affinity to these dyes. Cell death begins at stage 11 (approximately 7 hours) of embryogenesis and thereafter becomes widespread, affecting many different tissues and regions of the embryo. Although the distribution of dying cells changes drastically over time, the overall pattern of cell death is highly reproducible for any given developmental stage. Detailed analysis of cell death in the central nervous system of stage 16 embryos (13-16 hours) revealed asymmetries in the exact number and position of dying cells on either side of the midline, suggesting that the decision to die may not be strictly predetermined at this stage. This work provides the basis for further molecular genetic studies on the control and execution of programmed cell death in Drosophila.
The reaper gene (rpr) is important for the activation of apoptosis in Drosophila. To investigate whether rpr expression is sufficient to induce apoptosis, transgenic flies were generated that express rpr complementary DNA or the rpr open reading frame in cells that normally live. Transcription of rpr from a heat-inducible promoter rapidly caused widespread ectopic apoptosis and organismal death. Ectopic overexpression of
rpr in the developing retina resulted in eye ablation. The occurrence of cell death was highly sensitive to the dosage of the
transgene. Because cell death induced by the protein encoded by rpr (RPR) could be blocked by the baculovirus p35 protein, RPR appears to activate a death program mediated by a ced-3/ICE (interleukin-1 converting enzyme)-like protease.
In Drosophila, the chromosomal region 75C1-2 contains at least three genes, reaper (rpr), head involution defective (hid), and grim, that have important functions in the activation of programmed cell death. To better understand how cells are killed by these genes, we have utilized a well defined set of embryonic central nervous system midline cells that normally exhibit a specific pattern of glial cell death. In this study we show that both rpr and hid are expressed in dying midline cells and that the normal pattern of midline cell death requires the function of multiple genes in the 75C1-2 interval. We also utilized the P[UAS]/P[Gal4] system to target expression of rpr and hid to midline cells. Targeted expression of rpr or hid alone was not sufficient to induce ectopic midline cell death. However, expression of both rpr and hid together rapidly induced ectopic midline cell death that resulted in axon scaffold defects characteristic of mutants with abnormal midline cell development. Midline-targeted expression of the baculovirus p35 protein, a caspase inhibitor, blocked both normal and ectopic rpr- and hid-induced cell death. Taken together, our results suggest that rpr and hid are expressed together and cooperate to induce programmed cell death during development of the central nervous system midline.
CED-4 protein plays an important role in the induction of programmed cell death in Caenorhabditis elegans through the activation of caspases. However, the precise mechanisms by which it activates caspases remain unknown. To investigate the conservation of CED-4 function in evolution, transgenic Drosophila lines that express CED-4 in the compound eye were generated. Ectopic expression of CED-4 in the eyes induced massive apoptotic cell death through caspase activation. An ATP-binding site (P-loop) mutation in CED-4 (K165R) causes a loss of function in its ability to activate Drosophila caspase, and an ATPase inhibitor blocks the CED-4-dependent caspase activity in Drosophila S2 cells. Immunoprecipitation analysis showed that both CED-4 and CED-4 (K165R) bind directly to Drosophila caspase drICE, and the overexpression of CED-4 (K165R) inhibits CED-4-, ecdysone-, or cycloheximide-dependent caspase activation in S2 cells. Furthermore, CED-4 (K165R) partially prevented cell death induced by CED-4 in Drosophila compound eyes. Thus, CED-4 function is evolutionarily conserved in Drosophila, and the molecular mechanisms by which CED-4 activates caspases might require ATP binding and direct interaction with the caspases.
Macrophages in the Drosophila embryo are responsible for the phagocytosis of apoptotic cells and are competent to engulf bacteria. Croquemort (CRQ) is
a CD36-related receptor expressed exclusively on these macrophages. Genetic evidence showed thatcrq was essential for efficient phagocytosis of apoptotic corpses but was not required for the engulfment of bacteria. The expression
of CRQ was regulated by the amount of apoptosis. These data define distinct pathways for the phagocytosis of corpses and bacteria
in Drosophila.
Exposure of the aminophospholipid phosphatidylserine at the outer leaflet of the plasma membrane by apoptotic cells can trigger phagocytic removal of these dying cells. This functionality of phosphatidylserine exposure in the process of phagocytosis is indicated by in vitro studies of mammalian and insect phagocytes. We have studied the in vivo distribution of cell-surface exposed phosphatidylserine by injecting biotinylated Annexin V, a Ca2+ -dependent phosphatidyl-serine binding protein, into viable mouse and chick embryos and Drosophila pupae. The apparent binding of Annexin V to cells with a morphology which is characteristic of apoptosis and which was present in regions of developmental cell death indicates that phosphatidylserine exposure by apoptotic cells is a phylogenetically conserved mechanism.
Although insects lack adaptive immune systems endowed with memory elements and finely tuned discriminatory powers such as those found in the vertebrates, they do possess internal defense mechanisms for combating foreign materials (Salt, 1970). Among these are the cellular responses of phagocytosis and encapsulation used to resist parasitic and microbial infections. These processes do not involve opsonization of foreign materials (Scott, 1971; Anderson et al., 1973), so it appears that the insect hemocyte surfaces themselves must play the crucial role in discriminating nonself from self. This being the case, analysis of insect defense reactions requires study of hemocyte surface receptor sites on which extracellular cues operate as well as investigation of the body’s own tissue surfaces to which the hemocytes remain neutral.
Aqueous solutions of alcohol-acetic acid-formalin or glutaraldehyde-acrolein were shaken with heptane and heptane phase used for fixation. Phase-partition fixation is akin to fixation with vapor. The organic solvent, immiscible with water, penetrates hydrophobic membranes and carries the fixative in contact with water phase of the tissue. Only the fixative enters the tissue, without changing the ionic and water-soluble substance concentrations in the tissue. The quality of this fixation for optical or electron microscopy was as good as that of any conventional fixation method. Staining with basic fuchsin after 2 N HCl hydrolysis gave brilliant staining of nuclei, more intense than that with Feulgen reagent, while cytoplasm remained nearly colorless. Fixing and staining procedures for Drosophila eggs are given.
The embryonic lethal abnormal visual system (elav) gene of Drosophila melanogaster is required for the development and maintenance of the nervous system. Transcripts from this locus are distributed ubiquitously throughout the nervous system at all developmental stages. A product of this gene, the ELAV protein, has homology to known RNA binding proteins. The localization of the ELAV protein was studied in all developmental stages using antibodies that were generated against a hybrid protein made in Escherichia coli. In general, these data are consistent with previous results and demonstrate that (1) the ELAV protein is detected in the developing embryonic nervous system at a time coincident with the birth of the first neurons, (2) the ELAV protein is first detected in the majority of neurons of the central and peripheral nervous systems of embryos, larvae, pupae, and adults, (3) the ELAV protein appears to be localized to the nucleus, and (4) the ELAV protein is not detected in neuroblasts or identifiable glia. These data also provide new information concerning elav expression and show that (1) ELAV is not expressed in the ganglion mother cells (GMCs), (2) while the ELAV protein is localized to the nucleus, it is not uniformly distributed throughout this structure, and (3) other Drosophila species do express an ELAV-like antigen. We propose that the elav gene provides a neuronal-housekeeping function that is required for the successful posttranscriptional processing of transcripts from a set of genes the function of which is required for proper neuronal development and maintenance.
The regular, reiterated cellular pattern of the Drosophila compound eye makes it a sensitive amplifier of defects in cell death. Quantitative and histological methods reveal a phase of cell death between 35 and 50 h of development which removes between 2 and 3 surplus cells per ommatidium. The timing of this epoch is consistent with cell death as the last fate to be specified in the progressive sequence of cell fates that build the ommatidium. An ultrastructural survey of cell death suggests dying cells in the fly eye have similarities as well as differences with standard descriptions of programmed cell death.
A failure of cell death to remove surplus cells disorganizes the retinal lattice. A screen of rough eye mutants identifies two genes, roughest and echinus, required for the normal elimination of cells from the retinal epithelium.
The use of an enhancer trap as a cell lineage marker shows that the cone cells, like other retinal cells, are not clonally related to each other or to their neighbors.
The promoter from the metallothiortein gene may be a useful conditional promoter for the construction of chimeric genes to
be expressed in Drosophila cells in culture. To explore this possibility the responses of the endogenous metallothionein gene and an in vitro constructed
chimeric gene containing the metallothiortein promoter were examined. Copper and cadmium, when added to the growth medium
of Drosophila Schneider's line 2 cells, can produce a 30–100 fold induction of metallothionein mRNA levels. The level of induction depends
on the amount of copper or cadmium added to the medium and these mRNA levels remain high for at least four days. Copper is
less toxic than cadmium and does not induce a typical heat-shock response in the cells. Finally, a chimeric gene containing
the metallothionein promoter shows a similar induction when transformed into the cells.
Using an improved method of gel electrophoresis, many hitherto unknown
proteins have been found in bacteriophage T4 and some of these have been
identified with specific gene products. Four major components of the
head are cleaved during the process of assembly, apparently after the
precursor proteins have assembled into some large intermediate
structure.
At adult emergence, the ventral CNS of Drosophila shows a group of approximately 300 neurons, which are unique in that they express 10-fold higher levels of the A isoform of the ecdysone receptor (EcR-A) than do other central neurons. This expression pattern is established early in metamorphosis and persists throughout the remainder of the pupal stage. Although these cells represent a heterogeneous group of neurons, they all share the same fate of undergoing rapid degeneration after the adult emerges from the pupal case. One prerequisite for this death is the decline of ecdysteroids at the end of metamorphosis. Treatment of flies with 20-hydroxyecdysone blocks the death of the cells, but only if given at least 3 hours before the normal time of degeneration. The correlation of a unique pattern of receptor isoform expression with a particular steroid-regulated fate suggests that variations in the pattern of receptor isoform expression may serve as important switches during development.
The eyes absent (eya) gene is required at an early stage in development of the D. melanogaster compound eye. In eya mutants, progenitor cells in the eye disc undergo programmed cell death anterior to the morphogenetic furrow, rather than proceeding into the pathway of retinal differentiation. A low level of cell death normally occurs at this stage, suggesting that eya activity influences the distribution of cells between differentiation and death. Molecular analysis identifies a nuclear protein expressed in progenitor cells prior to differentiation. Transformation with the cDNA prevents progenitor cell death and allows the events that generate the eye to proceed. eya activity is required for the survival of eye progenitor cells at a critical stage in morphogenesis.
Programmed cell death is first observed at stage 11 of embryogenesis in Drosophila. The systematic removal of apoptotic cells is mediated by cells that are derived from the procephalic mesoderm and differentiate into macrophages. We describe a macrophage receptor for apoptotic cells. This receptor, croquemort (catcher of death), is a member of the CD36 superfamily. Croquemort-mediated phagocytosis represents the concept that phagocytosis evolved primarily as a cellular process for the removal of effete cells. Our findings support the idea that the primordial function of macrophages may have been in tissue modeling and that their adapted role is in host defense.
Activation of Ras inhibits apoptosis during Drosophila development. Genetic evidence indicates that Ras antiapoptotic activity in the developing eye is regulated by the Drosophila EGF receptor and operates through the Raf/MAPK pathway. Decreased activity of this pathway enhances, and increased activity suppresses, apoptosis induced by ectopic expression of the cell death regulators reaper (rpr) and head involution defective (hid). In addition, ectopic activation of the Ras/MAPK pathway in the developing embryo and in the developing eye suppresses naturally occurring apoptosis and regulates the transcription of the proapoptotic gene hid. Null alleles of hid recapitulate the antiapoptotic activities of Ras/MAPK, providing genetic evidence that downregulation of hid is an important mechanism by which Ras promotes survival.
We investigated the potential of double-stranded RNA to interfere with the function of genes in Drosophila. Injection of dsRNA into embryos resulted in potent and specific interference of several genes that were tested. In contrast, single-stranded RNA weakly interfered with gene activity. The method was used to determine the reception mechanism of the morphogen Wingless. Interference of the frizzled and Drosophila frizzled 2 genes together produced defects in embryonic patterning that mimic loss of wingless function. Interference of either gene alone had no effect on patterning. Epistasis analysis indicates that frizzled and Drosophila frizzled 2 act downstream of wingless and upstream of zeste-white3 in the Wingless pathway. Our results demonstrate that dsRNA interference can be used to analyze many aspects of gene function.
All of the following steps are done with gentle agitation on a platform shaker Programmed cell death during Drosophila embryogenesis
- Tunel
TUNEL Reaction (Reagents from Intergen Apoptag Kit) All of the following steps are done with gentle agitation on a platform shaker. References Kristin White et al. Abrams, J. M., White, K., Fessler, L. I., and Steller, H. (1993). Programmed cell death during Drosophila embryogenesis. Development 117, 29-43.
Characterization and spatial distribution of the ELAV protein during Drosophila melanogaster development Cell surface exposure of phosphatidylserine during apoptosis is phylogenetically conserved
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Robinow, S., and White, K. (1991). Characterization and spatial distribution of the ELAV protein during Drosophila melanogaster development. J. Neurobiol. 22(5), 443-461. van den Eijnde, S., Boshart, L., Baehrecke, E., De Zeeuw, C., Reutelingsperger, C., and Vermeij-Keers, C. (1998). Cell surface exposure of phosphatidylserine during apoptosis is phylogenetically conserved. Apoptosis 3, 9-16.