reaper
The genomic region containing reaper, grim, and head involution defective is required for all cell death in Drosophila embryos, including radiation-induced apoptosis. rpr is transcriptionally induced in embryos following irradiation, and an 11 kb sequence upstream of the rpr start codon is sufficient to confer
radiation responsiveness on a lacZ reporter transgene. To identify the minimal radiation-responsive cis-elements upstream of rpr,
the ability of smaller fragments of this 11 kb regulatory region to activate lacZ transcription was tested. Each transgenic strain was tested for
radiation-induced expression of beta-galactosidase. Multiple constructs
containing sequences ~5 kb upstream of the rpr start codon show a robust radiation response. These experiments
identify a discrete 150 bp enhancer
that responds to radiation as strongly as the larger enhancer fragments tested. Since this
enhancer retains radiation-responsiveness but does not recapitulate the developmental patterns of rpr expression seen with larger enhancer fragments, the results also indicate that cis-regulatory sequences responsible for damage-induced transcription of rpr can be isolated from
others that respond to developmental cues (Brodsky, 2000).
Within the 150 bp enhancer, a 20 bp sequence was identified that strongly resembles the consensus for human p53 DNA-binding sites. This 20 bp sequence is referred to as the p53 response element (p53RE) to reflect its response to Dmp53 in yeast. Like those found upstream
of the human target genes mdm-2 and p21/WAF1, this putative p53 binding site upstream of rpr contains two
tandemly arrayed 10mers, each of which matches the consensus motif at nine of ten positions. The two mismatches occur
at the outer positions of the 20 bp element; the invariant core nucleotides of each 10mer motif match the consensus perfectly (Brodsky, 2000).
Yeast one-hybrid assays were used to see whether Drosophila p53 interacts with the p53RE. For these studies, a
reporter plasmid containing the p53RE upstream of the beta-galactosidase gene was integrated into the yeast genome to produce the p53RE bait
strain. Next, this p53RE bait strain was transformed with test plasmids expressing either wild-type Dmp53 or Dmp53(259H) fused to the GAL4 activation
domain. These strains were assayed for beta-galactosidase activity. Reporter expression in strains with Dmp53(259H) or the empty
vector control (expressing the Gal4 activation domain alone) are indistinguishable from each other. Compared to these controls, each of the four independent
transformants carrying the wild-type Dmp53 plasmid shows a substantial increase in beta-galactosidase levels. Based on these results, it has been concluded
that the 150 bp radiation-responsive enhancer upstream of rpr contains a 20 bp binding site for Dmp53 (Brodsky, 2000).
A test was performed to see whether the p53RE is sufficient to confer radiation-responsive transcriptional activation on a lacZ reporter construct in vivo. A transgene
containing four copies of the p53RE and the minimal hsp70 promoter has showennegligible expression in untreated embryos but is substantially induced
following irradiation. Therefore, the 20 bp Dmp53 binding site from the rpr locus is sufficient to mediate a transcriptional response to radiation and
may define a minimal radiation responsive sequence. When analyzed in parallel to the 150-lacZ reporter, containing 150 bases surrounding the p53RE, the p53RE-lacZ reporter exhibits less robust and
less uniform beta-galactosidase activity following irradiation. Reduced activity is often observed when DNA elements are tested in
isolation from the normal flanking sequences and, in this instance, may reflect the influence of other factors that interact with the 150 bp enhancer sequence (Brodsky, 2000).
Disruptions of development are often associated with excess apoptosis. For example, in a crumbs (crb) mutant background,
abnormal epidermal development in the embryo leads to widespread apoptosis. This apoptosis is fully suppressed by deletions for the genomic region containing rpr, hid, and grim and is preceded by a dramatic induction of rpr
expression, similar to that seen in irradiated embryos. A test was performed to see whether transcriptional activation
mediated by p53RE represents a specific response to radiation damage or a common integration point for multiple pathways that lead to excess apoptosis.
Beta-galactosidase expression was examined in wild-type and crb embryos carrying either the p53RE-lacZ or the 2kb-lacZ reporter constructs. In stage 12/13 wild-type embryos, expression of the 2kb-lacZ transgene is normally confined to the developing gut but, in similarly aged crb
embryos, expression is induced throughout the epidermis. In contrast, the p53RE-lacZ transgene exhibits only basal expression in either wild-type or crb
embryos. Thus, despite widespread apoptosis in crb embryos, there is no induction of reporter expression from the p53RE. These results indicate
that the p53RE specifically responds to radiation damage, not generally to all proapoptotic signals. They also indicate that irradiation and disrupted
development may activate rpr expression through distinct pathways (Brodsky, 2000).
Hormones and trophic factors provide cues that control neuronal death during development. These
developmental cues in some way regulate activation of apoptosis, the mechanism by which most, if not
all, developmentally programmed cell deaths occur. In Drosophila, apoptosis can be induced by the
expression of the genes reaper, grim, or head involution defective. Prior to the
death of a set of identifiable doomed neurons, these neurons accumulate transcripts of the reaper and grim genes, but do not accumulate transcripts of the head involution defective gene. Death of these doomed neurons can be suppressed by two manipulations: either by increasing the levels of the steroid hormone 20-hydroxyecdysone (see Ecdysone receptor) or by decapitation. The impact that these two manipulations have on reaper expression has been investigated. Steroid treatment prevents the accumulation of reaper transcripts, whereas decapitation results in the accumulation of lower levels of reaper transcripts that are not sufficient to activate apoptosis. These data demonstrate that in vivo, reaper, and grim transcripts accumulate coordinately in a set of identified doomed neurons prior to the onset of apoptosis. These observations raise the possibility that products of the reaper and grim genes act in concert in postembryonic neurons to induce apoptosis. That reaper transcript accumulation is regulated
by the steroid hormone titer and by the presence of the head is evidence that developmental factors
control programmed cell death by regulating the expression of genes that induce apoptosis (Robinow, 1997).
Larval midgut and salivary gland histolysis are stage-specific steroid-triggered
programmed cell death responses. Larval salivary glands can be maintained for many hours in organ culture, providing an ideal opportunity to study the hormonal requirements for a variety of responses to ecdysone, including glue secretion, polytene chromosome puffing and specific gene regulation. The majority of glands cultured in the presence of ecdysone for 7 hours show a strong nuclear acridine orange stain indicating that salivary gland cell death is an ecdysone-triggered response. In vivo, dying larval midgut and salivary gland cell nuclei become permeable
to the vital dye acridine orange; their DNA undergoes fragmentation, indicative of apoptosis. Crawling mid-third instar larvae were injected with ecdysone. Such injected larvae pupariate within 6-8 hours, and midguts isolated from these larvae show a uniform nuclear acridine orange staining, indicative of the onset of programmed cell death.
The histolysis of these tissues can be inhibited by ectopic expression of the baculovirus
anti-apoptotic protein p35, implicating a role for caspases in the death response. Coordinate
stage-specific induction of the Drosophila death genes reaper (rpr) and head involution defective (hid)
immediately precedes the destruction of the larval midgut and salivary gland. The diap2
anti-cell death gene is repressed in larval salivary glands at the time that rpr and hid are induced, suggesting that the
death of this tissue is under both positive and negative regulation. diap2 is repressed by
ecdysone in cultured salivary glands under the same conditions that induce rpr expression and trigger
programmed cell death. These studies indicate that ecdysone directs the death of larval tissues via the
precise stage- and tissue-specific regulation of key death effector genes (Jiang, 1997).
The fact that tailless, brancyenteron and bowl expression at the blastoderm stage are all apparently normal in caudal-deficient embryos suggests that a hindgut primordium is established in the absence of cad activity. The lack of proper blastoderm stage expression of fkh and wg, however, indicates that this hindgut primordium is not properly specified. In byn mutant embryos one of the earliest phenotypic manifestations of an abnormally specified hindgut primordium is ectopic expression of the cell death gene reaper (rpr). To ask whether the extremely reduced hindgut in cad-deficient embryos might result from a similar course of programmed cell death, expression of rpr was examined in embryos lacking cad. A striking pattern of ectopic rpr expression is observed in cad minus embryos, beginning during stage 7 and continuing into stage 8 (gastrulation), in a ring at the circumference of the amnioproctodeal plate. However, the actual loss of cells that is presumably initiated by this ectopic rpr expression does not begin until after early stage 10, because the hindgut primordium is present at this stage in cad-deficient embryos, as indicated by its expression of byn and fkh. By stage 13, the cad-deficient embryo has a very short hindgut and no detectable anal pads; in sections of stage 13 embryos there are numerous apoptotic cells in the region of the hindgut remnant (Wu, 1998).
The steroid hormone ecdysone signals the stage-specific programmed cell death of the larval salivary glands during Drosophila metamorphosis. This response is
preceded by an ecdysone-triggered switch in gene expression in which the diap2 death inhibitor is repressed and the reaper (rpr) and head involution defective (hid) death activators are induced. rpr is induced directly by the ecdysone-receptor complex through an essential response element in the rpr
promoter. The Broad-Complex (BR-C) is required for both rpr and hid transcription, while E74A is required for maximal levels of hid induction. diap2 induction is
dependent on FTZ-F1, while E75A and E75B are each sufficient to repress diap2. This study identifies transcriptional regulators of programmed cell
death in Drosophila and provides a direct link between a steroid signal and a programmed cell death response (Jiang, 2000).
Although initial studies had indicated that rpr and hid are coordinately induced in the salivary glands approximately 12 hr after puparium formation, more recent work has shown that rpr is induced approximately 1.5 hr earlier than hid, suggesting that these death activators are regulated by distinct mechanisms. The timing of rpr induction is synchronous with the prepupal ecdysone pulse, suggesting that it may be induced as a primary response to the hormone, while the delay in hid induction suggests that it may be a secondary response to ecdysone. These two modes of regulation can be distinguished by their different sensitivity to the inhibition of protein synthesis. Salivary glands were dissected from 10 hr wild-type prepupae and cultured in insect medium supplemented with 20-hydroxyecdysone, either in the presence or absence of cycloheximide. Total RNA was extracted after 0, 2, or 4 hr of culture and analyzed by Northern blot hybridization. Both rpr and hid are induced within 2 hr of hormone treatment, consistent with the proposal that these genes are induced by ecdysone in late prepupal salivary glands. In the presence of the protein synthesis inhibitor cycloheximide, rpr transcription is both delayed and reduced, while hid expression is completely eliminated. These observations indicate that rpr is induced directly by the hormone-receptor complex, although maximal levels of rpr transcription also require the synthesis of ecdysone-induced proteins. In contrast, hid is induced solely as a secondary response to ecdysone. These observations are consistent with the timing of rpr and hid induction in staged salivary glands and provide a framework for defining the molecular mechanisms by which ecdysone regulates rpr and hid transcription, triggering salivary gland cell death (Jiang, 2000).
The BR-C is defined by three genetic functions: broad (br), reduced bristles on palpus (rbp), and l(1)2Bc. Earlier studies have shown that the rbp function of the BR-C is required for salivary gland cell death during metamorphosis. This result has been confirmed by finding that larval salivary glands are not destroyed by 22 hr after puparium formation in pupae that carry the rbp5 null allele. The high penetrance of this mutant phenotype suggests that rpr and hid may not be properly expressed in rbp5 mutant salivary glands (Jiang, 2000).
To test this hypothesis, salivary glands were dissected from staged rbp5 mutants, and rpr and hid expression was examined by Northern blot hybridization. Both rpr and hid transcription is significantly reduced in rbp5 mutant salivary glands, indicating that the failure of salivary gland cell death in this mutant can be attributed to its inability to express these death activators. Both betaFTZ-F1 and the ecdysone-inducible E93 early gene are expressed in rbp5 mutant salivary glands, indicating that the block in rpr and hid transcription is not simply due to developmental arrest of the mutant animals. BR-C is expressed in midprepupal salivary glands and thus would be present in the late prepupal glands used for the cycloheximide experiment described above. This explains why the reduced level of rpr transcription observed in the absence of protein synthesis is not as severe as the rbp5 mutant phenotype (Jiang, 2000).
Both molecular and genetic studies have indicated that the BR-C and E74 function together in common developmental pathways during the onset of metamorphosis. It was therefore asked whether, like the BR-C, E74 might contribute to the ecdysone-triggered destruction of larval salivary glands. In support of this proposal, salivary gland cell death is significantly delayed in E74P[neo] animals. This mutation is a null allele that inactivates the E74A promoter. While salivary glands in control animals are completely destroyed by 16 hr after puparium formation, approximately 20% of E74P[neo]Df(3L)st-81k19 animals have salivary glands at 24 hr after puparium formation. This partially penetrant cell death defect suggests that rpr and hid expression may be reduced in E74A mutant salivary glands. To test this hypothesis, salivary glands were dissected from staged E74P[neo]/Df(3L)st-81k19 mutants, and rpr and hid expression was examined by Northern blot hybridization. Although rpr transcription is unaffected by the E74P[neo] mutation, the levels of hid transcription are significantly reduced. This observation indicates that E74A is required for the maximal induction of hid but not rpr (Jiang, 2000).
The observation that rpr transcription is induced directly by ecdysone in cultured larval salivary glands indicates that one or more EcR/USP binding sites should be present in the rpr promoter. As a first step toward identifying these regulatory elements, the sequences required for ecdysone-inducible rpr transcription in larval salivary glands were mapped. 9.6 kb of the rpr promoter is sufficient to recapitulate certain aspects of the complex pattern of rpr expression during embryogenesis. Four P element constructs were made that carry either 9.5, 6.1, 3.9, or 1.2 kb of DNA upstream from the rpr transcription start site and 125 bp downstream from the transcription start site, with the rpr 5' untranslated region fused to a lacZ reporter gene. These constructs were introduced into the Drosophila germline by P element-mediated transformation, and the patterns of lacZ transcription in staged salivary glands were compared with those of the endogenous rpr gene by Northern blot hybridization. An increased level of rpr promoter activity is seen upon deletion of sequences between -9.5 and -6.1 kb relative to the start site of rpr transcription. The overall level of lacZ transcription is then reduced as more rpr regulatory sequences are deleted. However, 1.3 kb of the rpr promoter is sufficient to direct lacZ induction in synchrony with that of the endogenous rpr gene, indicating that this region contains the sequences required for proper temporal regulation (Jiang, 2000).
DNA fragments from the 1.3 kb rpr promoter region were generated by PCR and tested for their ability bind EcR: a 274 bp fragment extending from -195 bp to +80 bp relative to the rpr transcription start site binds EcR. Sequence analysis has shown a single imperfect palindromic EcR/USP binding site within this fragment. This rpr EcRE matches 10 out of 13 positions with the consensus EcR/USP binding site. The rpr element is not as strong of a binding site as a canonical hsp27 element. This observation is consistent with the deviations from the consensus at positions +2 and +3 in the rpr EcRE. These and other results strongly suggest that the ecdysone-receptor complex directly regulates rpr transcription through at least one binding site in the rpr promoter (Jiang, 2000).
Therefore ecdysone-regulated transcription factors encoded by betaFTZ-F1, BR-C, E74, and E75 function together to direct a burst of the diap2 death inhibitor followed by induction of the rpr and hid death activators. It is proposed that cooperation between rpr and hid allows these genes to overcome the inhibitory effect of diap2, by precisely coordinating when the salivary glands are destroyed. Evidence that the ecdysone-receptor complex directly induces rpr transcription through an essential response element in the promoter, providing a direct link between the steroid signal and a programmed cell death response. The diap2 death inhibitor is expressed briefly in the salivary glands of late prepupae, foreshadowing the imminent destruction of this tissue. This transient expression is directed by at least three ecdysone-regulated transcription factors: betaFTZ-F1, E75A, and E75B. diap2 induction is dependent on the betaFTZ-F1 orphan nuclear receptor. This is consistent with the timing of betaFTZ-F1 expression, which immediately precedes that of diap2, as well as the known role of betaFTZ-F1 as an activator of gene expression in late prepupae (Jiang, 2000).
Transgenic flies were examined in which immune deficiency (imd) expression was placed under the control of the ubiquitous da-GAL4 driver. It was surprising to observe 100% lethality in these flies during early larval development. The lethality was partially rescued by coexpression of the viral caspase inhibitor P35. This protein inactivates most of the executioner caspases of the death program. It was further noted that overexpression of imd by the fat body-specific driver yolk-GAL4 induced the transcription of a reaper-lacZ reporter in this tissue. Reaper is a key activator of apoptosis in Drosophila. A TUNEL analysis of transgenic flies overexpressing imd also revealed a remarkably large number of labeled nuclei in fat body cells, as compared to controls. This effect was suppressed by coexpression of the antiapoptotic protein P35. Transmission electron microscopy analysis revealed that the fat body cells exhibit the classical morphological aspects of apoptosis, that is, densification and fragmentation of the cytoplasm, membrane blebbing, and stacking of the endoplasmic reticulum (Georgel, 2001).
Steroid hormones trigger dynamic tissue changes during animal development by activating cell proliferation, cell differentiation, and cell death. Steroid regulation of changes have been characterized in midgut structure during the onset of Drosophila metamorphosis. Following an increase in the steroid 20-hydroxyecdysone (ecdysone) at the end of larval
development, future adult midgut epithelium is formed, and the larval midgut is rapidly destroyed. Mutations in the
steroid-regulated genes BR-C and E93 differentially impact larval midgut cell death but do not affect the formation of adult
midgut epithelia. In contrast, mutations in the ecdysone-regulated E74A and E74B genes do not appear to perturb midgut
development during metamorphosis. Larval midgut cells possess vacuoles that contain cellular organelles, indicating that
these cells die by autophagy. While mutations in the BR-C, E74, and E93 genes do not impact DNA degradation during this
cell death, mutations in BR-C inhibit destruction of larval midgut structures, including the proventriculus and gastric caeca,
and E93 mutants exhibit decreased formation of autophagic vacuoles. Dying midguts express the rpr, hid, ark, dronc, and
crq cell death genes, suggesting that the core cell death machinery is involved in larval midgut cell death. The transcription of rpr, hid, and crq are altered in BR-C mutants, and E93 mutants possess altered transcription of the caspase dronc, providing a mechanism for the disruption of midgut cell death in these mutant animals. These studies indicate that ecdysone triggers a two-step hierarchy composed of steroid-induced regulatory genes and apoptosis genes that, in turn, regulate the autophagic death of midgut cells during development (Lee, 2002).
Transcription of rpr, hid, ark, dronc, and crq increases in
wild-type animals following the late larval pulse of ecdysone
that triggers larval midgut cell death. Since
mutations in the BR-C and E93 genes prevent proper
destruction of larval midguts, Northern blots
were prepared from midguts of these mutants at stages
preceding and during cell death. BR-C 2Bc2 mutants have
altered transcription of rpr, hid, and crq, but do not impact
the transcription of ark and dronc. In contrast, E93
mutants possess altered transcription of dronc, but do not
change the transcript levels of the other cell death genes
known to be expressed in dying midguts. Although
midguts die by autophagy, they transcribe core apoptosis
regulators during this cell death, and mutants that prevent
autophagy alter transcription of apoptosis genes (Lee, 2002).
The distributed association of future adult cells within
the epithelium of larval midguts is another
important difference between ecdysone-regulated
midgut and salivary gland programmed cell death. The
close association of larval and adult midgut cells may be
one of the reasons why larval midgut exhibits a less
synchronized cell death than salivary glands. Both salivary
glands and midguts require the function of the E93 and
BR-C genes. However, mutations in these genes appear to
result in different effects in salivary glands and midguts;
BR-C appears to play a more important role in midguts. While both salivary glands and midguts express the cell
death genes rpr, hid, ark, dronc, and crq, the impact of
mutations in BR-C and E93 are very different in the midgut
than in salivary glands. BR-C affects transcription of rpr,
hid, and crq, but E93 mutants only affect dronc transcription
in midguts. In contrast, mutations in E93
prevent proper transcription of all of these cell death genes
in dying salivary glands. Clearly, many
more genes may be involved in the complicated autophagic
cell death of midguts. While several
similarities and differences have been identified between salivary gland and
midgut death, future analyses are needed to clarify the
mechanism by which the steroid ecdysone triggers midgut
programmed cell death (Lee, 2002).
Hox proteins control morphological diversity along the anterior-posterior body axis of animals, but the cellular processes these proteins regulate directly are poorly understood. During early Drosophila development, the Hox protein Deformed (Dfd) maintains the boundary between the maxillary and mandibular head lobes by activating localized apoptosis. Dfd accomplishes this by directly activating the cell death promoting gene reaper (rpr). One other Hox gene, Abdominal-B (Abd-B), also regulates segment boundaries through the regional activation of apoptosis. Thus, one mechanism used by Drosophila Hox genes to modulate segmental morphology is to regulate programmed cell death, which literally sculpts segments into distinct shapes. This and other emerging evidence suggests that Hox proteins may often regulate the maintenance of segment boundaries (Lohmann, 2002).
Several lines of evidence -- the effects of manipulating rpr expression in embryos, in vitro DNA binding studies with Dfd protein and mutagenesis of Dfd binding sites in the rpr enhancer, the phenocopy of the Dfd mutant boundary defect with an apoptosis inhibitory gene, its rescue with an apoptosis promoting gene, and the phenotype of rpr mutants -- show that the Hox protein Dfd is a direct transcriptional activator of rpr in the anterior maxillary segment, and that rpr expression and apoptosis are necessary to maintain the maxillary/mandibular boundary. At least in part, this Dfd-dependent, anterior maxillary expression of rpr is conferred by a 674 bp enhancer (rpr 4-S3) that maps 3.1 kb upstream of the rpr transcription start. This demonstrates that a Hox protein directly regulates a cell biological effector gene that mediates a morphological subroutine for that Hox function. Therefore, rpr qualifies as a directly regulated realizator. Interestingly, although Dfd is expressed in nearly all maxillary cells, the loss of Dfd function does not influence rpr expression in the posterior maxillary segment, indicating that other activators and/or repressors of rpr are distributed in maxillary cells that influence the transcriptional activity of Dfd protein on this locus. In the tail region, Abd-B is also required for the formation of normal boundaries between the abdominal segments A6/A7 and A7/A8, and their maintenance correlates, as in the case of the maxillary/mandibular boundary, with the localized activation of rpr. Thus, at both termini of the Drosophila body, Hox control of apoptosis is used for segment boundary maintenance (Lohmann, 2002).
Hox proteins may have a wider role in the programming of segmental boundaries than is currently believed. There is strong evidence that two Drosophila homeobox genes that are used to control segment number, even-skipped and fushi-tarazu, are independently derived from genes that still possess Hox segment identity functions in most insects and other arthropods. In addition, mutants in the mouse Hoxa-2 gene have segmentation defects in the hindbrain. Although a segmental boundary is normally established between rhombomeres 1 and 2 in the Hoxa-2 mutants, it is not maintained, which is reminiscent of the defect in boundary maintenance observed in Dfd mutant embryos (Lohmann, 2002).
Surprisingly, although rpr is required for maxillary/mandibular boundary maintenance during embryogenesis, flies lacking rpr function survive to adulthood with only minor defects. One possible explanation is that other cell death activators can compensate for the absence of rpr at later stages of development, since other apoptosis genes, like hid, grim, and sickle, are expressed in overlapping patterns with rpr and share IAP binding motifs in their N-terminal protein sequence. This may also explain why the maxillary/mandibular segmentation defect is less severe in Dfd null mutants and in XR38/H99 mutants when compared to homozygous Df(3L)H99 mutants. Since rpr and hid, but not grim, are expressed in anterior maxillary cells at many developmental stages, and since the combined activities of hid and rpr dictate the probability of a cell to undergo apoptosis, it is suggested that in wild-type embryos the combination of rpr and hid are required to kill cells at the maxillary/mandibular boundary (Lohmann, 2002).
Many Drosophila genes are known to be regulated in a Hox-dependent manner, but most encode either transcriptional regulators or cell signaling molecules. These Hox effectors presumably act both independently and/or in parallel to Hox genes to indirectly influence cell type identity and morphology. For example, the Hox target gene Distal-less (Dll) is required for the development of embryonic appendages and is directly repressed in abdominal segments by the Hox proteins Ubx and Abd-A. However, at some point the Hox proteins, their downstream effectors, and other cofactors must affect cellular changes by means of the class of realizer genes (Lohmann, 2002).
There are three good candidates for realizer genes in Drosophila: connectin, centrosomin, and ß-tubulin. connectin encodes an extracellular cell surface protein with leucine-rich repeats. It mediates cell-cell adhesion in cell culture assays and acts as a homophilic cell adhesion molecule in the lateral transverse muscles. In the nervous system, connectin expression is under the control of Ubx; a small regulatory fragment that mediates portions of connectin expression has been isolated by its affinity for Ubx in coimmunoprecitation assays. In some tissues, connectin is under the direct control of Ubx protein, but it is still unclear which of the morphogenetic subfunctions of Ubx require connectin function. centrosomin is a subunit of the centrosome and is necessary for the proper development of the CNS, PNS, and midgut. During the formation of the second midgut constriction, the functions of both Ubx and centrosomin are required, and centrosomin is lost in the visceral mesoderm cells of Ubx mutants. The ß-tubulin gene encodes a major component of microtubules and contains a cis-regulatory element that is regulated by Ubx in the visceral mesoderm (Lohmann, 2002).
In Drosophila, as in vertebrates, programmed cell death is used for the sculpting of morphological structures. For example, limb formation in amniotes is accompanied by massive cell death in almost all the interdigital mesenchymal tissue located between the chondrifying digits, eliminating the cells located between the differentiating cartilages and thus sculpting the shape of the limb. Interestingly, in Hoxa13 heterozygous mutant mice, the apoptosis that normally occurs in the interdigital regions is reduced, leading to a partial fusion of digits II and III in adult mice. In Hoxa13 homozygous mutant mice, there is no interdigital apoptosis and no digit separation in 14-day-old embryos. Although it remains to be seen whether Hoxa13 and other Hox genes are direct regulators of apoptotic genes in amniotes and other animals, one intriguing possibility is that Hox-dependent regulation of apoptosis is a more general mechanism used to generate and maintain metameric pattern during animal development (Lohmann, 2002).
An important issue in Metazoan development is to understand the mechanisms that lead to stereotyped patterns of programmed cell death. In particular, cells programmed to die may arise from asymmetric cell divisions. The mechanisms underlying such binary cell death decisions are unknown. A Drosophila sensory organ lineage is described that generates a single multidentritic neuron in the embryo. This lineage involves two asymmetric divisions. Following each division, one of the two daughter cells expresses the pro-apoptotic genes reaper and grim and subsequently dies. The protein Numb appears to be specifically inherited by the daughter cell that does not die. Numb is necessary and sufficient to prevent apoptosis in this lineage. Conversely, activated Notch is sufficient to trigger death in this lineage. These results show that binary cell death decision can be regulated by the unequal segregation of Numb at mitosis. This study also indicates that regulation of programmed cell death modulates the final pattern of sensory organs in a segment-specific manner (Orgogozo, 2002).
The vmd1a neuron is located within a cluster of five multidendritic (md) neurons in the ventral region of abdominal segments A1-A7. The vmd1a neuron can be distinguished from the other ventral md neurons (vmd1-4) using the B6-2-25 enhancer-trap marker. The origin of this vmd1a neuron is not known. vmd1-4 neurons are generated by the four vp1-4 external sensory (es) organ primary precursor (pI) cells. Each vp1-4 pI cell follows a lineage called the md-es lineage. This lineage is composed of four successive asymmetric cell divisions that generate five distinct cells, the four cells of the es organ at the position where the pI cell has formed and one md neuron that will then migrate to the ventral md cluster. In the md-es lineage, the membrane-associated protein Numb is segregated into one of the two daughter cells at each cell division. Numb establishes a difference in cell fate by antagonizing Notch in the Numb-receiving cell. Because no es organ is found in the vicinity of the vmd1a neuron, this neuron is probably not generated by a md-es lineage (Orgogozo, 2002).
rpr and grim, but not hid, are expressed specifically in the pIIa and pIIIb cells of the vmd1a lineage. By contrast, these genes are not expressed in cells of the vp1-4 lineages. In embryos in which a pIIb cell divides at the vp1 position in at least one abdominal segment, most segments contain a vmd1a pIIa-pIIb pair with one cell expressing rpr or grim. This cell is the pIIa cell fated to die. In some other segments, neither of these two cells accumulates rpr (25%) or grim (8%). Since the development of segments is not perfectly synchronous, it is assumed that this represents a situation preceding the onset of rpr and grim expression in the pIIa cell. In the remaining segments, a single Cut-positive cell is detected indicating that the pIIa cell has died. In those segments, expression of rpr and grim is never detected in the remaining pIIb cell (Orgogozo, 2002).
During the pIIb division, Numb was shown to segregate into the dorsal pIIb daughter cell. This cell is not fated to die and differentiates as a vmd1a neuron. By contrast, it could not be directly determined which one of the two pI daughter cells inherits Numb. Indeed, since the orientation of the vmd1a pI cell division is random, the pIIa and pIIb cells could not be identified from their relative positions. Nevertheless the vmd1a pIIa and pIIIb cells appear to generate ectopic shaft/socket and neuron/sheath cell pairs when cell death is prevented. In the md-es lineage, these cell pairs are the progeny of the cells that do not inherit Numb. This suggests that both the vmd1a pIIIb cell and the pIIa cell do not inherit Numb. Thus, Numb appears to segregate in the cells that do not die in the vmd1a lineage (Orgogozo, 2002).
The role of Numb was tested in regulating rpr and grim expression as well as cell death in the vmd1a lineage. In numb mutant embryos in which a secondary precursor cell divides at the vp1 position in at least one abdominal segment, it was observed that the two Cut-positive vmd1a pI daughter cells accumulate rpr or grim transcripts (54% of the segments for rpr, 52% for grim). In other segments a single Cut-positive pI daughter cell was found accumulating rpr or grim. In these segments one pI daughter cell has already died and the other one is undergoing apoptosis. These two phenotypes are not seen in wild-type embryos. Thus, in the absence of numb, both pI daughter cells undergo programmed cell death. Consistently, no Cut-positive cell is observed at the vmd1a position in numb mutant embryos in most segments. It is concluded that numb is required to inhibit the expression of rpr and grim and to prevent cell death in the pIIb cell (Orgogozo, 2002).
To test whether numb is sufficient to prevent cell death, the progeny of the vmd1a pI cell was analyzed in arm-Gal4 UAS-numb embryos that express high levels of Numb. In wild-type embryos in which a vp1 pIIIb cell is dividing in at least one segment, one or two Cut-positive cells are observed at the vmd1a position. In contrast, four Cut-positive cells are observed in 50% of the segments in arm-Gal4 UAS-numb embryos at the same stage. In 8 out of the 9 segments with four cells, two cells accumulating high levels of Pros and two cells accumulating low levels of Pros are seen, suggesting that these cells are two vmd1a neurons and two pIIIb cells. These data indicate that the pIIa cell death is inhibited and that the pIIa cell is transformed into a pIIb-like cell (Orgogozo, 2002).
Numb is known to function by antagonizing Notch activity. This therefore suggests that Notch promotes cell death in the vmd1a lineage and that Numb blocks this activity of Notch. Unfortunately, the strong effect of Notch loss-of-function alleles on the selection of the vmd1a pI cell means that it was not possible to test directly whether Notch is required for cell death in the vmd1a lineage. Therefore the conditional Notchts1 allele was used. However, when Notchts1 embryos are shifted to a restrictive temperature (31°C) soon after the specification of the vmd1a pI cell (i.e., at 13-14.5 hours after egg laying at 19°C), no significant reduction was seen in the number of rpr- or grim-expressing pIIa cells. A stronger Notchts1/Notch55e11 combination causes the appearance of additional vmd1a pI cells even at the permissive temperature (19°C). It is therefore not possible to determine whether an increase in the number of rpr- or grim-negative cells results from a lack of Notch-dependent apoptosis or from an excess of vmd1a pI cells due to reduced Notch signaling during lateral inhibition (Orgogozo, 2002).
Therefore a test was performed to see whether an activated form of Notch, Nintra, can promote the death of the pIIb cell when expressed around the time of the vmd1a pI cell division. In 6% of the segments from embryos in which at least one segment shows a dividing vp1 pIIb cell, rpr or grim transcripts accumulate in both vmd1a pI daughter cells. In other segments, a single Cut-positive cell remains at the vmd1a position and accumulates rpr or grim. These expression patterns are not seen in heat-shocked control embryos. Importantly, these observations are similar to those made in numb mutant embryos. Thus, both loss of numb activity and ectopic Notch signaling lead to transcriptional activation of pro-apoptotic genes in the pIIb cell. Finally, a similar effect of Nintra on rpr and grim expression is seen in the vmd1a pIIb daughter cells when Nintra expression was induced at a later stage, i.e., when the vmd1a pIIb cell is dividing. Together, these results indicate that Notch signaling is sufficient to promote cell death in the vmd1a lineage (Orgogozo, 2002).
In summary, the lineage generating the vmd1a neuron has been described. This lineage is composed of two asymmetric divisions following which one daughter cell undergoes apoptosis. These two binary cell death decisions are regulated by the unequal segregation of Numb at mitosis. Therefore, the data provide the first experimental evidence that alternative cell death decision can be regulated by the unequal segregation of a cell fate determinant. The conserved role of Numb and Notch in neuronal specification in flies and vertebrates suggests that Numb-mediated inhibition of Notch may play a similar role in regulating cell death decisions in vertebrates (Orgogozo, 2002).
In vertebrates, neurons often undergo apoptosis after differentiating and
extending their axons. By contrast, in the developing nervous system of invertebrate embryos apoptosis typically occurs soon after cells are generated. The Drosophila dMP2 and MP1 pioneer neurons undergo segment-specific apoptosis at late embryonic stages, long after they have extended their axons and have performed their pioneering role in guiding follower axons. This segmental specificity is achieved by differential expression of the Hox gene Abdominal B, which in posterior segments prevents pioneer neuron death postmitotically and cell-autonomously by repressing the RHG-motif cell death activators reaper and grim. These results identify the first clear case of a cell-autonomous and anti-apoptotic role for a Hox gene in vivo. In addition, they provide a novel mechanism linking Hox positional information to differences in neuronal architecture along the anteroposterior axis by the selective elimination of mature neurons (Miguel-Aliaga, 2004).
How does Abd-B prevent the function of RHG-motif genes? It is likely that Abd-B prevents pioneer neuron apoptosis by repressing the transcription of, at least, rpr and grim. This idea is supported by four facts: (1) the H99 deletion is epistatic to (functions downstream of) Abd-B; (2) Abd-B is a transcription factor; (3) rprGAL4 is activated posteriorly in Abd-Bm mutants; (4) when misexpressed postmitotically, Abd-B can fully rescue both types of pioneer neurons. Given that loss of rpr is critical for anterior dMP2 survival, whereas loss of grim is critical for anterior MP1s, Abd-B must prevent the expression of at least these two cell death activators (Miguel-Aliaga, 2004).
In the developing vertebrate neural tube, a number of studies have shown that Hox genes are critical for AP organization and for proper neuronal specification. Although their action may be largely confined to progenitor cells, recent studies have revealed that Hox genes can also act to control the identity of early postmitotic neurons. In the light of the current findings, it will be of interest to determine if selective, Hox-dependent elimination of mature neurons gives rise to differences in motor neuron numbers along the AP axis of the vertebrate spinal cord. Increased apoptosis of postmitotic motor neurons has been observed in mouse mutants lacking Hoxc-8, one of the vertebrate homologues of abd-A. This may be the result of the aberrant connectivity pattern of Hoxc-8-deficient motor neurons, which would restrict their access to target-derived neurotrophic factors. However, this increase in cell death is also consistent with the possibility that Hoxc-8 normally acts to prevent apoptosis of postmitotic neurons in its expression domain (Miguel-Aliaga, 2004).
The results contrast with the previous finding that Abd-B appears to activate rpr transcription to regulate segment boundary formation in the posterior region of early Drosophila embryos. Decreased apoptosis has also been observed in mouse mutants lacking Hoxb13, one of the vertebrate homologues of Abd-B. It has previously been shown that the target functions of Hox genes are highly dependent on cellular context, and the regulation of apoptosis appears to be no exception. This context dependence may not be unique to the Abd-B gene. abd-A has been previously reported to activate apoptosis in post-embryonic neuroblasts during normal development. When Antp and Ubx were misexpressed in these neuroblasts, they too were able to trigger apoptosis. In contrast, none of these genes acted in a pro-apoptotic manner in the current study. It is, therefore, conceivable that the pro-apoptotic function of Hox genes is confined to progenitors, at least in the nervous system. Alternatively, or additionally, availability of certain cofactors may determine whether a Hox gene activates or represses transcription of pro-apoptotic genes in a specific cell (Miguel-Aliaga, 2004).
In addition to their dependence on cellular context, specific Hox proteins may control pro-apoptotic genes differently. Abd-B and its vertebrate homologues share several properties that distinguish them from other Hox proteins, such as the absence of a hexapeptide motif and a preference for a different DNA core sequence. Together, these differences may confer unique transcriptional properties on proteins of the Abd-B family, and may explain why Abd-B is the only Hox protein capable of fully rescuing anterior pioneer neurons. The finding that Abd-B is the only Hox gene that was unable to rescue the embryonic brain phenotypes of Drosophila mutants for the Hox gene labial is consistent with this idea (Miguel-Aliaga, 2004).
Is the cellular control of Hox gene expression functionally relevant? The results show that while Hox genes are broadly expressed within their domains, they are largely absent from certain cell populations; at stage 16, few glial cells express Hox genes in the VNC. Since many Drosophila neuroblasts give rise to both neurons and glia, it is possible that Hox gene expression is actively suppressed by factors promoting glial fate. Alternatively, an initial wave of Hox expression in progenitors could be followed by a second, neuron-specific re-activation of Hox expression. In any case, it will be of interest to identify the molecular mechanism by which Hox gene expression is confined to specific populations of postmitotic cells in the nervous system (Miguel-Aliaga, 2004).
While cellular context may determine whether a Hox gene acts in a pro- or anti-apoptotic manner, apoptosis of specific cells within a Hox expression domain may also be achieved by differential Hox gene expression. For example, while Abd-A is broadly expressed in abdominal segments during larval stages, it is absent from post-embryonic neuroblasts. However, at the last larval instar, a neuroblast-specific pulse of abd-A results in the activation of the cell death program in these cells. Similarly, and given the novel role for Hox proteins in the apoptosis and differentiation of postmitotic neurons, the expression of Hox genes in specific postmitotic neurons is likely to be of functional significance. Together, these findings are not consistent with the view that Hox genes solely function as 'segment identity' factors specifying global properties of the segments in which they are active. Instead, they lend functional support to the proposal that Hox genes are required for a number of decisions taken at the cellular level (Miguel-Aliaga, 2004).
The combined activity of RHG-motif genes is critical to the initiation of all cell death in the Drosophila embryo. These genes act in an additive manner. However, not all cell death activators are simultaneously expressed in every cell fated to die, and their specific expression patterns do not always overlap. Therefore, it is likely that they are differentially regulated by specific developmental signals. While Abd-B acts to repress rpr and grim function in posterior pioneer neurons, the developmental stimulus activating their expression in these neurons throughout the cord is currently unknown. Three developmental signals are known to regulate the function of RHG-motif genes in the Drosophila nervous system. The insect hormone ecdysone appears to be important for blocking cell death of certain peptidergic neurons during metamorphosis. However, the ecdysone-receptor complex has also been shown to promote cell death by activating rpr transcription in other tissues during Drosophila metamorphosis. While an embryonic ecdysone pulse occurs around the time when pioneer neurons die, preliminary experiments have failed to lend any support to an ecdysone-dependent activation of apoptosis in these neurons. The EGF-receptor/Ras/MAPK pathway has been shown to phosphorylate Hid protein, thereby preventing apoptosis of midline glial cells. However, neither Rpr nor Grim appear to be regulated in this fashion, and this model would not address the specific transcriptional activation of these genes in pioneer neurons. Lastly, Notch signaling has been described as resulting in both activation and inhibition of apoptosis. In Drosophila, recent studies have revealed that Notch can act cell-autonomously to induce apoptosis during final mitotic divisions both in the central and peripheral nervous systems. Although this Notch-induced developmental apoptosis is prevented in H99 mutant embryos, the molecular mechanisms by which activated Notch signaling results in the activation of IAP inhibitors are still unknown. Nevertheless, Notch signaling is unlikely to be relevant to dMP2 death, since it is not active in dMP2 neurons. It is, therefore, likely that an as yet unidentified factor is responsible for the activation of the apoptotic machinery in pioneer neurons. This factor could be Odd-skipped, given its specific expression in dMP2 and MP1 neurons. Because of the early role of odd in embryonic patterning, its possible postmitotic function in these neurons cannot be addressed using the currently available odd mutants (Miguel-Aliaga, 2004).
Developmental apoptosis in invertebrate embryos typically occurs shortly after cells are generated. In Drosophila, this has often precluded the identification of dying cells until apoptosis has been genetically prevented. Consequently, progress in identification of the mechanisms controlling apoptosis has been relatively slow, and little is known about the upstream pathways that initiate cell death in specific tissues or lineages. Furthermore, in the Drosophila VNC, studies have shown that apoptotic corpses are engulfed by glia, transported to the dorsal surface of the VNC and transferred to macrophages for final destruction. The molecular genetic mechanisms underlying this intriguing series of events are only just beginning to be unraveled. The identification of a late apoptotic event in two of the best-studied and least complex lineages in the Drosophila CNS, as well as the characterization of the dMP2-GAL4 line, should contribute to the elucidation of the mechanisms involved in both the developmental initiation and execution of apoptosis (Miguel-Aliaga, 2004).
To discover whether expression of apoptosis activators reaper, grim and hid triggers the accumulation of Death related ced-3/Nedd2-like protein (DREDD) mRNA, the three apoptosis activators were ectopically expressed in mesoderm, and the expression of DREDD mRNA examined. Expression of the apoptosis activators triggers excessive apoptosis in mesoderm. During stage 13 and beyond, DREDD mRNA is not widely expressed in the developing musculature in wild-type flies. However, when misexpression of each of the death activators is directed to these tissues, prominent levels of ectopic DREDD mRNA are detected. Expression of grim in the ectoderm also results in DREDD mRNA accumulation. DREDD mRNA accumulation has also been examined in embryos homozygous for crumbs (crb). In crb mutants, reaper is ectopically expressed in the disorganized epidermis. As anticipated, ectopic accumulation of DREDD mRNA is found scattered throughout the ectoderm in crb embryos, coincident with widespread patterns of rpr expression. Perhaps the most compelling evidence for a direct role for Dredd in apoptosis comes from an examination of accumulation of DREDD mRNA in embryos carrying a homozygous deletion of the entire reaper region (mutated for rpr, hid, and grim). No apoptosis occurs in these deletion mutants. The selective accumulation of DREDD mRNA fails to occur in these mutants. This is the first report of a molecular activity that is completely blocked by the absence of H99-associated signaling (Chen, 1998).
Drosophila affords a genetically well-defined system to study apoptosis in vivo. It offers a powerful
extension to in vitro models that have implicated a requirement for cytochrome c in caspase activation
and apoptosis. An overt alteration in cytochrome c anticipates programmed cell death
(PCD) in Drosophila tissues, occurring at a time that considerably precedes other known indicators of
apoptosis. The altered configuration is manifested by display of an otherwise hidden epitope and occurs
without release of the protein into the cytosol. Conditional expression of the Drosophila death
activators, reaper or grim, provoke apoptogenic cytochrome c display and, surprisingly, caspase
activity is necessary and sufficient to induce this alteration. In cell-free studies, cytosolic caspase
activation is triggered by mitochondria from apoptotic cells but identical preparations from healthy
cells are inactive. These observations provide compelling validation of an early role for altered
cytochrome c in PCD and suggest propagation of apoptotic physiology through reciprocal,
feed-forward amplification involving cytochrome c and caspases (Varkey, 1999).
Two cytochrome c genes, DC4 and DC3, have been described in Drosophila studies at the level of protein and at the level
of RNA that suggest that DC4 (which shows >86% identity with its rat
counterpart) is either the predominant or only form of actively expressed product. An
existing panel of mAbs, directed against mammalian versions of cytochrome c, was screened in a search for possible probes for in
situ analyses of the fly counterpart. Two mAbs, 6H2 and 2G8, recognize Drosophila cytochrome c.
Both antibodies detected a doublet that comigrates with mammalian cytochrome c at ~13 kD. While
mAb 2G8 preferentially precipitates the upper band, mAb 6H2 has about equal affinity for both forms
of cytochrome c. No obvious correlation between the relative abundance of the two cytochrome c
bands and apoptosis is observed.
These bands clearly represent distinct cytochrome c species. Although the biochemical nature of these different forms
is unresolved, mAb 2G8 preferentially recognizes the higher molecular weight
form of the doublet. This product occurs in relatively small amounts that are not overtly affected by
the extent of apoptosis in the cultures (Varkey, 1999).
Between stages 11 and 13 of Drosophila oogenesis, programmed cell death eliminates nurse cells
which nourish the developing egg. The apoptotic nature of nurse cell death is indicated by two distinct markers:
acridine orange staining and TUNEL labeling.
These readily identifiable cells offer a unique opportunity to examine pre-apoptotic events before their
eventual demise. To directly demonstrate the involvement of cytochrome c during apoptosis, Drosophila ovaries were
stained with the anti-cytochrome c mAbs described above and egg chambers at all stages of
development were analyzed. Only nurse cells at stage
10B exhibit pronounced cytochrome c immunoreactivity distributed as characteristically punctate
labeling of the cytoplasm in a pattern consistent with localization to mitochondria. To determine the chronology of cytochrome c display, relative to other apoptotic changes,
the onset of mAb binding was compared with other degenerative changes known to occur in these cells. Though nurse
cells at stage 10B show pronounced exposure of the epitope for mAb 2G8, no signs of
apoptosis are apparent in either the cytoplasm or the nuclei of these cells. By
stages 12-13 (at least 0.5 to 4 h later) the nuclei of the dying nurse cells adopt characteristic apoptotic
features as evidenced by the TUNEL assay and acridine orange staining.
These results demonstrate that cytochrome c display precedes overt signs of apoptosis in intact organs (Varkey, 1999).
Previous studies on Drosophila SL2 cells have shown that conditional expression of rpr or grim
triggers apoptosis in cultured cells and in transgenic animals. Transiently transfected SL2 cells were induced for
rpr or grim and, at various time intervals after induction, the preparations were examined for
cytochrome c immunoreactivity with mAb 2G8. Apoptotic cultures
exhibit profound staining with the antibody. To test the possibility that cytochrome c might be released into the cytosol during apoptosis, healthy SL2 cells and apoptotic rpr- or grim-expressing cells were fractionated, and assayed for cytochrome
c in the mitochondrial and cytosolic fractions. Surprisingly, these cells showed no
difference in cytochrome c distribution and no evidence was found for the transit of cytochrome c to the
cytosol as a correlate to apoptosis. Biochemical data indicating retention of cytochrome c in mitochondria during apoptosis is consistent
with cytological studies. These observations indicate that
appreciable efflux of cytochrome c from mitochondria does not occur during apoptosis in Drosophila
cells (Varkey, 1999).
Mitochondria isolated from apoptotic cells trigger caspase activation in vitro.
Caspase activation was measured in L2 cell cytosol that had been coincubated with mitochondria isolated
from parental L2 cells or from pre-apoptotic cells (induced either for rpr or grim). Caspase activation was detected, as measured by signature cleavage of a bovine substrate, PARP.
Cleavage of PARP in this assay is indistinguishable from the signature activity
reported in many mammalian systems and is readily detected in the cytosol of pre-apoptotic cells but not in cytosol from parental L2. These observations
emphasize the importance of one or more mitochondrial factors in the activation of caspase function
triggered by rpr or grim (Varkey, 1999).
The Drosophila death activators, rpr and grim, activate one or more caspases to elicit apoptosis. To study the temporal relation of cytochrome c display with respect to caspase activity, SL2 cells were cotransfected with rpr and p35 plasmids. Six hours after induction, cells induced for rpr alone
show pronounced labeling with mAb 2G8 whereas cells expressing rpr together with p35
are prevented from apoptosis and do not bind the mAb. These observations suggest that
apoptogenic cytochrome c display requires caspase activity, a presumption that is further
substantiated when rpr-expressing cells are treated with the peptide caspase inhibitors zDEVD-fmk
and zVAD-fmk. As seen for p35-blocked cells, these inhibitors similarly prevent mAb 2G8 labeling
and subsequent apoptosis. Parallel results are observed in grim-expressing cells
(Varkey, 1999).
These data demonstrate that caspase activity is required for apoptogenic cytochrome c display. To
determine if caspase function is sufficient to trigger this change, apoptosis was induced in SL2 cells by
conditional expression of an activated version of the Drosophila caspase, dcp-1. If deleted for its
prodomain, this caspase provokes considerable apoptosis in mammalian cells
and SL2 cells. When labeled with mAb 2G8, cells transfected and induced for dcp-1 expression exhibit profound punctate cytochrome c staining with
features indistinguishable from those associated with expression of the death activators (Varkey, 1999).
Two potential explanations reconcile the in vivo observations reported here on apoptogenic cytochrome c with
reports from mammalian cell-free systems that cytochrome c can trigger caspase activation. One
possibility is that the order and/or nature of cytochrome c apoptotic function is not conserved between
mammals and insects and thus, relative to caspase action, cytochrome c is upstream in the former case
and downstream in the latter case. This scenario, however, seems unlikely given the widespread
conservation of apoptotic components, the fact that display of fly cytochrome c in the animal
significantly precedes all signs of programmed cell death, and reports from mammalian systems that
upstream caspases can trigger cytochrome c release. Therefore, a more likely interpretation of the results reported here is that
cytochrome c propagates apoptotic physiology by functioning together with caspases in a feed-forward
amplification loop. In this scenario, altered cytochrome c and caspase activity exert positive and
reciprocal feedback on one another, similar to observations recently reported for caspase 8. Thus, agents that restrain caspase action (p35) are also predicted to suppress
pro-apoptotic display of cytochrome c, which behaves as an amplifier of caspase function. This
interpretation is also consistent with recent studies on Fas signaling in type II cells, where molecular
ordering studies found that activation of an initiator caspase (caspase 8/Flice) occurs upstream of
changes associated with cytochrome c (Varkey, 1999).
Inhibitor of apoptosis (IAP) proteins suppress apoptosis and inhibit caspases. Several IAPs also function as ubiquitin-protein ligases. Regulators of IAP auto-ubiquitination, and thus IAP levels, have yet to be identified. Head involution defective (Hid), Reaper (Rpr) and Grim downregulate
Drosophila melanogaster IAP1 (DIAP) protein levels. Hid stimulates DIAP1
polyubiquitination and degradation. In contrast to Hid, Rpr and Grim can
downregulate DIAP1 through mechanisms that do not require DIAP1 function as a
ubiquitin-protein ligase. Observations with Grim suggest that one mechanism by which these proteins produce a relative decrease in DIAP1 levels is to promote a general suppression of protein translation. These observations define two mechanisms through which DIAP1 ubiquitination controls cell death: first, increased ubiquitination promotes degradation directly; second, a decrease in global protein synthesis results in a differential loss of short-lived proteins such as DIAP1. Because loss of DIAP1 is sufficient to promote caspase activation, these mechanisms should promote apoptosis (Yoo, 2002).
Inhibitors of apoptosis (IAPs) inhibit caspases, thereby preventing proteolysis
of apoptotic substrates. IAPs occlude the active sites of caspases to which they
are bound and can function as ubiquitin ligases. IAPs are also reported to
ubiquitinate themselves and caspases. Several proteins induce apoptosis, at
least in part, by binding and inhibiting IAPs. Among these are the Drosophila
melanogaster proteins Reaper (Rpr), Grim, and HID, and the mammalian proteins
Smac/Diablo and Omi/HtrA2, all of which share a conserved amino-terminal
IAP-binding motif. Rpr not only inhibits IAP function, but
also greatly decreases IAP abundance. This decrease in IAP levels results from a combination of increased IAP degradation and a previously unrecognized ability of Rpr to repress total protein translation. Rpr-stimulated IAP degradation required both IAP ubiquitin ligase activity and an unblocked Rpr N terminus. In contrast, Rpr lacking a free N terminus still inhibits protein translation. Since the abundance of short-lived proteins are severely affected after translational inhibition, the coordinated dampening of protein synthesis and the ubiquitin-mediated destruction of IAPs can effectively reduce IAP levels to lower the threshold for apoptosis (Holley, 2002).
The Drosophila reaper, head involution defective, and grim genes play key roles in regulating the activation of programmed cell death. Two useful systems for studying the functions of these genes are the embryonic CNS midline and adult eye. The Gal4/UAS targeted gene expression system has been used to demonstrate that unlike reaper or hid, expression of grim alone is sufficient to induce ectopic CNS midline cell death. In both the midline and eye, grim-induced cell death is not blocked by the Drosophila anti-apoptosis protein Diap2, which does block both reaper- and hid-induced cell death. grim can also function synergistically with reaper or hid to induce higher levels of midline cell death than observed for any of the genes individually. Finally the function was analyzed of a truncated Reaper-C protein that lacks the NH2-terminal 14 amino acids that are conserved between Reaper, Hid, and Grim. Ectopic expression of Reaper-C reveals cell killing activities distinct from full length Reaper, and indicates that the conserved NH2-terminal domain acts in part to modulate Reaper activity (Wing, 2001a).
Morphological hallmarks of apoptosis result from activation of the caspase family of cysteine proteases, which are opposed by a pro-survival family of inhibitors of apoptosis proteins (IAPs). In Drosophila, disruption of IAP function by Reaper, HID, and Grim (RHG) proteins is sufficient to induce cell death. RHG proteins have been reported to localize to mitochondria, which, in the case of both Reaper and Grim proteins, is mediated by an amphipathic helical domain known as the GH3. Through direct binding, Reaper can bring the Drosophila IAP (DIAP1) to mitochondria, concomitantly promoting IAP auto-ubiquitination and destruction. Whether this localization is sufficient to induce DIAP1 auto-ubiquitination has not been reported. This study characterized the interaction between Reaper and the mitochondria using both Xenopus and Drosophila systems. Reaper concentrates are found on the outer surface of mitochondria in a nonperipheral manner largely mediated by GH3-lipid interactions. Importantly, mitochondrial targeting of DIAP1 alone is not sufficient for degradation and requires Reaper binding. Conversely, Reaper is able to bind IAPs, but lacking a mitochondrial targeting GH3 domain (DeltaGH3 Reaper), can induce DIAP1 turnover only if DIAP1 is otherwise targeted to membranes. Surprisingly, targeting DIAP1 to the endoplasmic reticulum instead of mitochondria is partially effective in allowing DeltaGH3 Reaper to promote DIAP1 degradation, suggesting that co-localization of DIAP and Reaper at a membrane surface is critical for the induction of DIAP degradation. Collectively, these data provide a specific function for the GH3 domain in conferring protein-lipid interactions, demonstrate that both Reaper binding and mitochondrial localization are required for accelerated IAP degradation, and suggest that membrane localization per se contributes to DIAP1 auto-ubiquitination and degradation (Freel, 2008).
MicroRNAs are small noncoding RNAs that control gene function posttranscriptionally through mRNA degradation or translational inhibition. Much has been learned about the processing and mechanism of action of microRNAs, but little is known about their biological function. Injection of 2′O-methyl antisense oligoribonucleotides (2'OM-ORNs) into early Drosophila embryos leads to specific and efficient depletion of microRNAs and thus permits systematic loss-of-function analysis in vivo. Twenty-five of the forty-six embryonically expressed microRNAs show readily discernible defects; pleiotropy is moderate and family members display similar yet distinct phenotypes. Processes under microRNA regulation include cellularization and patterning in the blastoderm, morphogenesis, and cell survival. The largest microRNA family in Drosophila (miR-2/6/11/13/308) is required for suppressing embryonic apoptosis; this is achieved by differential posttranscriptional repression of the proapoptotic factors hid, grim, reaper, and sickle. These findings demonstrate that microRNAs act as specific and essential regulators in a wide range of developmental processes (Leaman, 2005).
miR-2/13 and miR-6 depletion results in catastrophic apoptosis: Embryos injected with miR-2/13 and miR-6 antisense 2′OM-ORNs fail to differentiate normal internal and external structures. At the end of embryogenesis, the embryos fall apart on touch, and no cuticle is recovered. To determine the onset of these problems, blastoderm embryos were examined, and it was found that cellularization and early pattern formation along the anteroposterior axis occur normally for both miRNAs, indicating that early fating and morphogenesis are intact. Interestingly, in miR-6, but not miR-2/13 depleted embryos, pole cell formation at the posterior end is disrupted (Leaman, 2005).
One possible cause of the catastrophic defects observed in miR-2/13 and miR-6 depleted embryos is excessive and widespread apoptosis. In both miR-2/13 and miR-6 antisense injected embryos, the number of apoptotic cells is greatly increased compared to wild-type by stage 13. Notably, the overall morphology of miR-6 depleted embryos is much more affected than that of miR-2/13 depleted embryos. miR-6 depleted embryos are generally smaller in size and have fewer and abnormally large (para-) segments, suggesting greater excess or earlier onset of apoptosis (Leaman, 2005).
To determine the specificity of the effects of miR-6 and miR-2/13 antisense injections, genomic rescue experiments were carried out. Embryos ubiquitously overexpressing mir-6 or mir-2 (Actin-Gal4;UAS-mir6-3/2b-2) show normal cell-death patterns. When injected with miR-6 or miR-2/13 antisense, they show significant rescue of miR-6 antisense by mir-6, with respect to both cell death and morphology, and of miR-2/13 antisense by mir-2. Interestingly, crossrescue of miR-6 antisense by mir-2 overexpression and of miR-2/13 antisense by mir-6 is weak (Leaman, 2005).
The miRNA sequence family miR-6 and miR-2/13 belong to has two additional members, miR-11 and miR-308. Depletion of miR-11 results in a moderate and of miR-308 in a mild increase in apoptosis in midembryogenesis. Thus, for all members of the miR-2 family, antisense-induced depletion results in excess embryonic cell death, but with marked differences in phenotypic strength. This differential could be due to differences in expression level or to sequence divergence and thus differential interaction with target mRNAs (Leaman, 2005).
The miR-2 family regulates cell survival by translational repression of proapoptotic factors: In Drosophila, three pathways are known to control caspase activity. The main control is thought to come from the proapoptotic factors Hid, Grim, and Reaper (Rpr), which are transcriptionally activated in response to a range of natural and toxic conditions; they promote caspase activation through inhibition of the caspase inhibitor Diap1. The three factors appear to act independently, with each being sufficient to drive apoptosis. When miR-2/13 and miR-6 antisense 2′OM-ORNs are injected into embryos deficient for the hid, grim, and rpr genes (H99 deficiency), they are unable to trigger apoptosis, indicating that these miRNAs act through hid, grim, and/or rpr (Leaman, 2005).
To determine whether the regulation of the three proapoptotic factors occurs at the transcriptional or at the posttranscriptional level, their RNA expression was examined in miR-2/13 and miR-6 depleted embryos using in situ hybridization and quantitative PCR. No significant increase was found in the expression level or broadening of the pattern compared to control embryos for any of three transcripts, either at embryonic stage 13 or 1 hr earlier at embryonic stage 12. By contrast, the protein expression of Hid is dramatically increased in miR-6 depleted embryos and modestly in miR-2/13 depleted embryos. These results strongly argue against a transcriptional and in favor of a posttranscriptional regulation of the proapoptotic factors by miR-2/13 and miR-6 (Leaman, 2005).
To test this directly, two existing translation control assays were adapted to the embryonic paradigm. In the first assay, full-length 3′UTRs are fused to a ubiquitously transcribed sensor (tub-GFP); transgenic embryos are injected with sense or antisense 2′OM-ORNs, and GFP fluorescence is measured. The 3′UTRs of hid, grim, rpr, and sickle (skl, a structurally related but less potent proapoptotic factor display marked differences in sensor expression, with rpr showing no expression, hid and skl low uniform expression, and grim strong and spatially modulated expression, indicating that these proapoptotic factors experience quite different levels of translation control. To gauge the efficacy of the assay, hid GFP sensor embryos were injected with bantam antisense 2′OM-ORNs, and mild but statistically significant derepression of GFP expression was found as compared to control, consistent with the weak cell-death phenotype of bantam depleted embryos. Antisense injection of miR-2 family members reveals strong derepression of the hid GFP sensor by miR-6 antisense, but not by miR-2/13, 11, or 308 antisense. Conversely, the grim GFP sensor shows significant derepression as a result of miR-2/13, 11, and 308, but not miR-6 depletion. Finally, the skl GFP sensor shows significant derepression for all four family members (Leaman, 2005).
To assess effects on rpr, a second, more sensitive assay was developed that employs transient expression of a dual-luciferase vector in injected embryos. For initial comparison with the GFP assay, a hid luciferase sensor was tested against the entire miR-2 family and the same profile was found. The rpr luciferase sensor shows strong derepression in miR-6 and 2/13, moderate derepression in miR-11, and no significant effect in miR-308 depleted embryos. Thus, the 3′UTRs of all four proapoptotic factors are subject to translational repression by the miR-2 family, but each miRNA displays a distinct interaction profile. The interaction preferences correlate well with the observed differences in phenotype: miR-6 has the most severe death phenotype and is the only family member to regulate hid, the factor with the broadest expression and the strongest proapoptotic effect. mir-2/13 and miR-11 have the same overall profile, but they differ in the strength of their interaction with rpr and show a corresponding differential in phenotypic strength. Finally, miR-308, which has the mildest death phenotype, interacts only with the weakly proapoptotic skl and with grim (Leaman, 2005).
The differences in target interaction profile between the miR-2 family members are pronounced and do not merely reproduce differences in the strength or onset of miRNA expression. This suggests that differential pairing outside the 5′ core sequence shared by all members has an important role in target selection. Computational predictions indicate that miR-2 family binding sites are present in the 3′UTRs of all four proapoptotic factors: rpr and grim have one, hid and skl two predicted sites. All six miRNA target sites lie in sequence blocks that are conserved between the six sequenced Drosophilid species, spanning an evolutionary distance of 40 Myr. Interestingly, for all sites, absolute conservation extends well beyond the bases complementary to the 5′ core of the miRNA and includes adjacent stretches suitable for pairing with the 3′ end. All but one of the sites show Watson-Crick pairing with miRNA positions 2-7 and variable pairing at the 3′ end. One of the hid sites (hid468) has a mismatch in the core but shows strong pairing with miR-6 at the 3′ end. The rules for 3′ pairing between miRNAs and their targets are not yet well understood, but it is clear that the miR-2 family members differ considerably in their ability to form 3′ matches with the six target sites. Further experimentation will be required to better understand how the observed differences in regulatory effect relate to differences in sequence pairing (Leaman, 2005).
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