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).

Epigenetic blocking of an enhancer region controls irradiation-induced proapoptotic gene expression in Drosophila embryos

Drosophila embryos are highly sensitive to gamma-ray-induced apoptosis at early but not later, more differentiated stages during development. Two proapoptotic genes, reaper and hid, are upregulated rapidly following irradiation. However, in post-stage-12 embryos, in which most cells have begun differentiation, neither proapoptotic gene can be induced by high doses of irradiation. The sensitive-to-resistant transition is due to epigenetic blocking of the irradiation-responsive enhancer region (IRER), which is located upstream of reaper but is also required for the induction of hid in response to irradiation. This IRER, but not the transcribed regions of reaper/hid, becomes enriched for trimethylated H3K27/H3K9 and forms a heterochromatin-like structure during the sensitive-to-resistant transition. The functions of histone-modifying enzymes Hdac1(Rpd3) and Su(var)3-9 and PcG proteins Su(z)12 and Polycomb are required for this process. Thus, direct epigenetic regulation of two proapoptotic genes controls cellular sensitivity to cytotoxic stimuli (Zhang, 2008).

Irradiation responsiveness appears to be a highly conserved feature of reaper-like IAP antagonists. A recently identified functional ortholog of reaper in mosquito genomes, michelob_x (mx), is also responsive to irradiation. These results highlighted that stress responsiveness is an essential aspect of functional regulation of upstream proapoptotic genes such as reaper/hid. It is also worth mentioning that several mammalian BH3 domain-only proteins, the upstream proapoptotic regulators of the Bcl-2/Ced-9 pathway, are also regulated at the transcriptional level (Zhang, 2008).

This study shows that the irradiation responsiveness of reaper and hid is subject to epigenetic regulation during development. The epigenetic regulation of the IRER is fundamentally different from the silencing of homeotic genes in that the change of DNA accessibility is limited to the enhancer region while the promoter of the proapoptotic genes remains open. Thus, it seems more appropriate to refer this as the 'blocking' of the enhancer region instead of the 'silencing' of the gene. This region, containing the putative P53RE and other essential enhancer elements, is required for mediating irradiation responsiveness. ChIP analysis indicates that histones in this enhancer region are quickly trimethylated at both H3K9 and H3K27 at the sensitive-to-resistant transition period, accompanied by a significant decrease in DNA accessibility. DNA accessibility in the putative P53RE locus (18,368k), when measured by the DNase I sensitivity assay, did not show significant decrease until sometime after the transition period. It is possible that other enhancer elements, in the core of IRER_left, are also required for radiation responsiveness. An alternative explanation is that the strong and rapid trimethylation of H3K27 and association of PRC1 at 18,366,000-18, 368,000 are sufficient to disrupt DmP53 binding and/or interaction with the Pol II complex even though the region remains relatively sensitive to DNase I. Eventually, the whole IRER is closed with the exception of an open island around 18,387,000 (Zhang, 2008).

The finding that epigenetic regulation of the enhancer region of proapoptotic genes controls sensitivity to irradiation-induced cell death may have implications in clinical applications involving ionizing irradiation. It suggests that applying drugs that modulate epigenetic silencing may help increase the efficacy of radiation therapy. It also remains to be seen whether the hypersensitivity of some tumors to irradiation is due to the dedifferentiation and reversal of epigenetic blocking in cancer cells. In contrast, loss of proper stress response to cellular damage is implicated in tumorigenesis. The fact that the formation of heterochromatin in the sensitizing enhancer region of proapoptotic genes is sufficient to convey resistance to stress-induced cell death suggests it could contribute to tumorigenesis. In addition, it could also be the underlying mechanism of tumor cells' evading irradiation-induced cell death. This is a likely scenario given that it has been well documented that oncogenes such as Rb and PML-RAR fusion protein cause the formation of heterochromatin through recruiting of a human ortholog of Su(v)3-9. In this regard, the reaper locus, especially the IRER, provides an excellent genetic model system for understanding the cis- and trans-acting mechanisms controlling the formation of heterochromatin associated with cellular differentiation and tumorigenesis (Zhang, 2008).

The developmental consequence of epigenetic regulation of the IRER is the tuning down (off) of the responsiveness of the proapoptotic genes, thus decreasing cellular sensitivity to stresses such as DNA damage. Epigenetic blocking of the IRER corresponds to the end of major mitotic waves when most cells begin to differentiate. Similar transitions were noticed in mammalian systems. For instance, proliferating neural precursor cells are extremely sensitive to irradiation-induced cell death while differentiating/differentiated neurons become resistant to γ-ray irradiation, even though the same level of DNA damage was inflicted by the irradiation. These findings here suggest that such a dramatic transition of radiation sensitivity could be achieved by epigenetic blocking of sensitizing enhancers (Zhang, 2008).

Later in Drosophila development, around the time of pupae formation, the organism becomes sensitive to irradiation again, with LD50 values similar to what was observed for the 4–7 hr AEL embryos. Interestingly, it has also been found that during this period, the highly proliferative imaginal discs are sensitive to irradiation-induced apoptosis, which is mediated by the induction of reaper and hid through P53 and Chk2. However, it remains to be studied whether the reemergence of sensitive tissue is due to reversal of the epigenetic blocking in the IRER or the proliferation of undifferentiated stem cells that have an unblocked IRER (Zhang, 2008).

The blocking of the IRER differs fundamentally from the silencing of homeotic genes in several aspects. (1) The change of DNA accessibility and histone modification is largely limited to the enhancer region. The promoter regions of reaper (and hid) remain open, allowing the gene to be responsive to other stimuli. Indeed, there are a few cells in the central nervous system that could be detected as expressing reaper long after the sensitive-to-resistant transition. Even more cells in the late-stage embryo can be found having hid expression. Yet, the irradiation responsiveness of the two genes is completely suppressed in most if not all cells, transforming the tissues into a radiation-resistant state (Zhang, 2008).

(2) The histone modification of the IRER has a mixture of features associated with pericentromeric heterochromatin formation and canonic PcG-mediated silencing. Both H3K9 and H3K27 are trimethylated in the IRER. Both HP1, the signature binding protein of the pericentromeric heterochromatin, and PRC1 are bound to the IRER. As demonstrated by genetic analysis, the functions of both Su(var)3-9 and Su(z)12/Pc are required for the silencing. Preliminary attempts to verify specific binding of PRC2 proteins to this region were unsuccessful. The fact that none of the mutants tested could completely block the transition seems to suggest that there is a redundancy of the two pathways in modifying/blocking the IRER. It is also possible that the genes tested are not the key regulators of IRER blocking but only have participatory roles in the process (Zhang, 2008).

(3) Within the IRER, there is a small region around 18,386,000 to 18,188,000 that remains relatively open until the end of embryogenesis. Interestingly, this open region is flanked by two putative noncoding RNA transcripts represented by EST sequences. If they are indeed transcribed in the embryo as suggested by the mRNA source of the cDNA library, then the 'open island' within the closed IRER will likely be their shared enhancer/promoter region. Sequences of both cDNAs revealed that there is no intron or reputable open reading frame in either sequence. Despite repeated efforts, their expression was not confirmed via ISH or northern analysis. Overexpression of either cDNA using an expression construct also failed to show any effect on reaper/hid-induced cell death in S2 cells. Yet, sections of the two noncoding RNAs are strongly conserved in divergent Drosophila genomes. The potential role of these two noncoding RNAs in mediating reaper/hid expression and/or blocking of the IRER remains to be studied (Zhang, 2008).

Coordinated expression of cell death genes regulates neuroblast apoptosis

Properly regulated apoptosis in the developing central nervous system is crucial for normal morphogenesis and homeostasis. In Drosophila, a subset of neural stem cells, or neuroblasts, undergo apoptosis during embryogenesis. Of the 30 neuroblasts initially present in each abdominal hemisegment of the embryonic ventral nerve cord, only three survive into larval life, and these undergo apoptosis in the larvae. This study used loss-of-function analysis to demonstrate that neuroblast apoptosis during embryogenesis requires the coordinated expression of the cell death genes grim and reaper, and possibly sickle. These genes are clustered in a 140 kb region of the third chromosome and show overlapping patterns of expression. Expression of grim, reaper and sickle in embryonic neuroblasts is controlled by a common regulatory region located between reaper and grim. In the absence of grim and reaper, many neuroblasts survive the embryonic period of cell death and the ventral nerve cord becomes massively hypertrophic. Deletion of grim alone blocks the death of neuroblasts in the larvae. The overlapping activity of these multiple cell death genes suggests that the coordinated regulation of their expression provides flexibility in this crucial developmental process (Tan, 2011).

The patterns of developmental cell death are extremely complex and dynamic, and the fate of an individual cell probably represents the integration of multiple signaling pathways. Intrinsic pathways that define the identity and health of a cell, as well as extrinsic growth and survival pathways, are all likely to contribute to the life or death decision. In Drosophila, the transcriptional regulation of the RHG genes is an important output of the pathways that regulate cell death. The clustering of these genes to a single large genomic locus suggests that cell death signals can be integrated by regulatory sequences that control the coordinated expression of these genes (Tan, 2011).

This work shows that rpr, grim and skl are co-regulated to eliminate NBs during embryonic development. A genomic region important for this regulation was identified, located between rpr and grim. Identified genes or transcripts are surprisingly lacking in the 93 kb region between these genes, which is highly conserved between Drosophila species. This suggests that crucial cis-regulatory elements might be localized to this region (Tan, 2011).

The genomic region important for NB apoptosis was narrowed down to 22 kb. The number of NBs expressing grim and skl is significantly decreased in stage 14/15 embryos homozygous for a deletion of this region. The number of NBs expressing rpr is also affected, but to a smaller extent. Interestingly, there is still expression of grim, rpr and skl in NBs from stage 10 through stage 16. However, in the wild type, the number of NBs expressing all three genes increases at stage 14, presaging the loss of these cells by several hours. This increase in NBs expressing rpr, grim and skl is not seen in MM3 homozygous embryos. As overall grim, rpr and skl expression levels are not significantly altered in the mutants, the deleted regulatory sequences must control grim, rpr and skl expression in both a temporal- and tissue-specific manner, and baseline NB expression must be regulated by sequences outside of this region (Tan, 2011).

It is also interesting to note that the MM3 deletion has a more significant effect on grim and skl expression than on rpr expression. grim and skl are co-expressed in most cells of the stage 15 embryonic CNS, whereas rpr expression is less overlapping. As grim and skl lie 134 kb apart, on either side of rpr, long range enhancer interactions might regulate subsets of genes in this cell death locus. This type of long-range interaction has been implicated previously in the response to irradiation. Here again, a single regulatory region regulates multiple cell death genes at a great distance. The radiation responsive element activates hid and rpr, which are 200 kb apart, without having a major effect on the intervening gene grim. It will be important to examine higher order chromatin interactions to understand these long-range enhancer interactions (Tan, 2011).

The structure of the RHG cell death locus is conserved in other Drosophila species, but not obviously in more distantly related insect species, even though IAP inhibitory proteins are found in other species. This suggests that a coordinately regulated RHG gene cluster might be particularly important for Drosophila development (Tan, 2011).

As demonstrated by these studies, the individual roles for the cell death genes in developmental apoptosis are not well understood. Strong genetic and molecular data support a role for hid in regulating cell death in the embryonic head and in the developing eye. A role for grim in the death of microchaete glial cells has been described recently (Wu, 2010). Shown here is a specific role for grim in the elimination of the normal abdominal neuroblasts in the third instar larvae. As described in this work, rpr alone is not required for abdominal NB death in the embryo. However, this double mutant analysis demonstrates that rpr and grim must act together to eliminate these cells (Tan, 2011).

A possible role for hid in NB death cannot be eliminated, although no hid mRNA is detected in the VNC outside of the midline. A recent study suggests that rpr requires hid for efficient induction of apoptosis (Sandu, 2010). However, loss of hid does not result in increased NB survival, as would be expected if hid is required for rpr activity. Furthermore, loss of one copy of hid along with rpr and grim does not increase NB survival. Knockdown of rpr, grim and hid in NBs with an RHG miRNA also does not increase the number of surviving NBs over loss of rpr and grim. Further studies are needed to examine how hid and grim, rpr and skl differentially contribute to developmental apoptosis (Tan, 2011).

Although skl has similar pro-apoptotic activity to hid, grim and rpr, a role for skl in developmental apoptosis has not previously been demonstrated, owing to the lack of a skl mutation. This study shows that skl deletion alone does not alter NB death. In addition, it was found that deletion of one copy of skl along with grim and rpr slightly increase NB survival over deletion of grim and rpr alone. Deletion of skl and rpr, along with the NBRR in XR38/X20 also results in a larger VNC than deletion of rpr and the NBRR in XR38/H88, again suggesting a role for skl in cell death in this tissue. Additional double mutant analysis will be needed to test whether deletion of skl along with grim or rpr supports a role for skl in developmental apoptosis. Given the strongly overlapping expression of grim and skl in the embryonic VNC, it might be found that skl is redundant with grim in the CNS (Tan, 2011).

What are the developmental advantages of multiple cell death regulators? This work demonstrates that multiple RHG genes are activated within a single NB to activate apoptosis. In the salivary gland, hid and rpr, but not grim, are transcribed in the dying tissue, whereas the radiation response appears to involve hid, rpr and skl. These data suggest that the complex upstream regulation of cell death is likely to impact on different subsets of the RHG genes. If each gene is controlled by different combinations of enhancers this could provide greater flexibility in controlling the activation of cell death. In addition, a requirement for multiple cell death activators could ensure that cells are not killed inappropriately by the accidental activation of a single RHG gene (Tan, 2011).

The requirement for multiple RHG genes to kill cells might also reflect differences in the pro-apoptotic activities of these genes. For example, the RHG proteins show differential binding preferences to specific domains of the DIAP1 protein, and inhibit DIAP1 anti-apoptotic functions through different mechanisms. Additional activities, such as mitochondrial permeabilization, translational repression or interaction with other proteins such as Bruce or Scythe, also differ between RHG genes. Furthermore, post-translational regulation of particular RHG proteins might regulate their activity. This has been demonstrated for Hid; phosphorylation by MAPK downregulates the killing activity of Hid. It remains to be demonstrated whether different cell types have differential sensitivities to particular combinations of cell death genes when expressed at normal levels. This might provide yet another layer of control of the cell death process (Tan, 2011).

In sum, this work demonstrates that the regulation of NB apoptosis during development involves the coordinated temporal and spatial regulation of multiple cell death genes, controlled by a common regulatory region. By dissecting the RHG gene locus, specific genomic elements that regulate these genes in other developmentally important cell deaths are likely to be identified (Tan, 2011).

Transcriptional regulation

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).

A steroid-triggered transcriptional hierarchy controls salivary gland cell death during Drosophila metamorphosis

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).

Drosophila Immune deficiency (IMD) is a death domain protein that activates antibacterial defense and can promote apoptosis

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 regulation of midgut cell death during Drosophila development

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).

The Drosophila Hox gene Deformed sculpts head morphology via direct regulation of the apoptosis activator reaper

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).

Binary cell death decision regulated by unequal partitioning of Numb at mitosis

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).

Segment-specific prevention of pioneer neuron apoptosis by cell-autonomous, postmitotic Hox gene activity

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).

Apical deficiency triggers JNK-dependent apoptosis in the embryonic epidermis of Drosophila

Epithelial homeostasis and the avoidance of diseases such as cancer require the elimination of defective cells by apoptosis. This study investigated how loss of apical determinants triggers apoptosis in the embryonic epidermis of Drosophila. Transcriptional profiling and in situ hybridisation show that JNK signalling is upregulated in mutants lacking Crumbs or other apical determinants. This leads to transcriptional activation of the pro-apoptotic gene reaper and to apoptosis. Suppression of JNK signalling by overexpression of Puckered, a feedback inhibitor of the pathway, prevents reaper upregulation and apoptosis. Moreover, removal of endogenous Puckered leads to ectopic reaper expression. Importantly, disruption of the basolateral domain in the embryonic epidermis does not trigger JNK signalling or apoptosis. It is suggested that apical, not basolateral, integrity could be intrinsically required for the survival of epithelial cells. In apically deficient embryos, JNK signalling is activated throughout the epidermis. Yet, in the dorsal region, reaper expression is not activated and cells survive. One characteristic of these surviving cells is that they retain discernible adherens junctions despite the apical deficit. It is suggested that junctional integrity could restrain the pro-apoptotic influence of JNK signalling (Kolahgar, 2011).

This study has shown that apical, but not basolateral, disruption leads to apoptosis in a developing epithelium. It was also showed that JNK signalling is a key intermediate in the signal transduction mechanism that triggers apoptosis in response to the loss of apical determinants. Apical disruption leads to activation of JNK signalling, which in turn activates transcription of the pro-apoptotic gene rpr. Moreover, rpr expression is not activated in apically disrupted embryos that are prevented from activating JNK signalling. Interestingly, in bicoid-deficient embryos and other segmentation mutants, cell fate misspecification requires activation of a different pro-apoptotic gene, hid. Therefore, distinct quality control pathways might exist to ensure that different forms of defective cells are removed from developing epithelia. JNK signalling has been shown to mediate apoptosis in a variety of other situations, including after DNA damage. However, JNK signalling does not necessarily cause apoptosis. Indeed, this pathway modulates many other cell activities, such as proliferation, differentiation and morphogenesis. What conditions determine whether JNK triggers apoptosis or not is an important issue. Another obvious question raised by the current findings concerns the nature of the mechanism that triggers JNK signalling following the loss of apical determinants (Kolahgar, 2011).

This study has shown that, in the embryonic epidermis, JNK signalling is activated by the loss of apical, not basolateral, determinants. In fact, reduction of lgl activity prevents JNK activation in crb mutant embryos. Similarly, Scrib knockdown prevents JNK activity in the mouse mammary epithelium, suggesting that the loss of the basolateral domain could have a general anti-JNK (and perhaps anti-apoptotic) activity. Although JNK activation has been documented in tissues that lack a basolateral determinant, it is suggested that this might be an indirect consequence of cell competition, which triggers JNK signalling, or of the specific experimental conditions (partial reduction of Puc activity). Overall, the results suggest the existence of an apical domain-dependent activity that modulates JNK signalling in the embryonic epidermis of Drosophila. This activity could be similar to that postulated to be at work in cultured MDCK cells, but is likely to be distinct from the process that leads to apoptosis in response to mosaic disruption of the basolateral domain in imaginal discs (Kolahgar, 2011).

The mechanism underlying the activation of JNK signalling by loss of apical determinants remains unknown. For example, it is uncertain at this point whether there is an apically localised activity that directly modulates JNK signalling or whether a more indirect route is at work (paths 1 and 2 respectively; see Signalling upstream and downstream of JNK). Since apical organisation is required for the establishment of adherens junctions, it is conceivable that the effect of apical disruption on JNK signalling is mediated by junctional disruption. This possibility is compatible with the absence of ectopic JNK activation in basolateral mutants, in which E-cadherin remains localised to patches at the cell surfac. However, one would have to invoke that slight junctional disruption is sufficient to trigger JNK signalling, as this pathway is upregulated in the dorsal region of crb mutants, where the extent of junctional disruption is relatively mild. It has not been possible to discriminate between paths 1 and 2, partly because of the current difficulty in eliminating adherens junctions from early Drosophila embryos. Future work will require novel means of interfering specifically with adherens junctions. Considering the lack of involvement of Egr, it will also be necessary to identify the upstream components of JNK signalling that respond to epithelial disruption (Kolahgar, 2011).

Overexpression of Puc, a feedback inhibitor of JNK signalling, prevents apoptosis in the ventral epidermis of crb embryos. This is clear evidence that JNK signalling is required for apical deficit to trigger apoptosis. However, it is well established that JNK signalling does not necessarily lead to apoptosis. This is particularly well illustrated by the situation at the dorsal edge of wild-type embryos, where JNK is highly active without triggering apoptosis. Moreover, in crb mutants, a 6- to 10-cell-wide band of dorsal tissue is refractory to the pro-apoptotic influence of JNK signalling. Therefore, additional conditions must be met for JNK signalling to activate rpr expression and trigger apoptosis. This study has identified two situations when refractory cells succumb to the pressure of JNK signalling (see Signalling upstream and downstream of JNK). One involves the reduction of Puc and the other the removal of zygotic E-cadherin activity. The first situation suggests that endogenous Puc can limit the ability of JNK to activate rpr expression and trigger apoptosis. The important role of Puc in preventing cell death is also highlighted by the extensive apoptosis seen in embryos lacking both maternal and zygotic Puc activity. Puc could act solely by limiting the extent of JNK signalling, thus preventing the very high level of signalling required for rpr expression. Alternatively, or in addition, Puc could have an activity that specifically prevents certain genes, such as rpr, from being spuriously activated. In any case, it is likely that the regulatory relationships between JNK, Puc and apoptosis are influenced by the cellular context (e.g., the state of adherens junctions (Kolahgar, 2011).

JNK signalling triggers rpr expression (and apoptosis) more readily if adherens junctions are weakened or disrupted. Therefore, junctional integrity could also protect epithelial cells from the pro-apoptotic effects of JNK signalling. Dorsoventral differences in junctional integrity and remodelling have been noted in the embryonic epidermis of Drosophila and these might explain why these two regions respond differently to the loss of crb. The results suggest that residual junctional integrity in the dorsal epidermis prevents JNK signalling from activating rpr expression. It is conceivable that a protective signal emanates from adherens junctions. Alternatively, junctional disruption could interfere with the ability of Puc to rein in the effect of JNK signalling on rpr expression. Although differential junctional dynamics between the dorsal and ventral epidermis could determine the propensity to undergo apoptosis, the possibility cannot be excluded that other dorsoventral determinants are at work too (Kolahgar, 2011).

This study has shown that loss of apical polarity leads to apoptosis by activating JNK signalling and causing junctional disruption. It is expected that this response, which is readily detectable in the crb mutant condition, might reflect a process that ensures the removal of abnormal and damaged cells during epithelial homeostasis. It is hoped that understanding the machinery that links epithelial disruption to JNK signalling and the transcription of pro-apoptotic genes will suggest means of reactivating this pathway in pathological situations (Kolahgar, 2011).

The cis-regulatory code of Hox function in Drosophila

Precise gene expression is a fundamental aspect of organismal function and depends on the combinatorial interplay of transcription factors (TFs) with cis-regulatory DNA elements. While much is known about TF function in general, understanding of their cell type-specific activities is still poor. To address how widely expressed transcriptional regulators modulate downstream gene activity with high cellular specificity, binding regions were identified for the Hox TF Deformed (Dfd) in the Drosophila genome. This analysis of architectural features within Hox cis-regulatory response elements (HREs) shows that HRE structure is essential for cell type-specific gene expression. It was also found that Dfd and Ultrabithorax (Ubx), another Hox TF specifying different morphological traits, interact with non-overlapping regions in vivo, despite their similar DNA binding preferences. While Dfd and Ubx HREs exhibit comparable design principles, their motif compositions and motif-pair associations are distinct, explaining the highly selective interaction of these Hox proteins with the regulatory environment. Thus, these results uncover the regulatory code imprinted in Hox enhancers and elucidate the mechanisms underlying functional specificity of TFs in vivo (Sorge, 2012).

In order to quantitatively identify genomic regions bound by the Hox TF Dfd in Drosophila, two complementing approaches were employed: ChIP-seq, which has been successfully applied previously to identify stage- and tissue-specific enhancer activities, and computational detection of clusters of TF binding sequences, which allows the identification of cis-regulatory modules irrespective of temporal and spatial context. To generate genome-wide maps of Dfd binding in vivo, ChIP was performed using stage 10-12 Drosophila embryos and a Dfd-specific antibod. Stage-independent in silico Dfd-specific Hox response elements (HREs) were identified by searching for clusters of conserved Dfd binding motifs, as defined by a position weight matrix (PWM), in the non-coding regions of the genomes of 12 distinct Drosophila species. By applying both approaches, 4526 genomic regions containing clusters of Dfd binding sites and 1079 Dfd ChIP-seq enrichment peaks were identified, including two out of the three well-characterized Dfd-HREs, namely rpr-4S3 and Dfd-EAE. To study the regulatory capacity of novel in silico and ChIP-seq detected HREs, cell culture-based enhancer assays were performed for 11 randomly selected HREs, and it was found that reporter expression driven by the identified genomic regions was in all cases dependent on Dfd binding. In vivo activity was tested of 21 arbitrarily selected enhancers in transgenic reporter lines, revealing that 7 out of 11 ChIP-identified and 5 out of 10 in silico-predicted Dfd-HREs recapitulate the spatio-temporal expression of adjacent genes). Most importantly, it was possible to demonstrate Dfd-dependent regulation of both transgenic reporter expression and endogenous gene expression, suggesting that they are bona fide direct Dfd target genes. Thus, the identified Dfd-HREs represent a data set of biologically relevant regulatory regions and an excellent resource to unravel sequence features within Hox responsive enhancers that might be essential for the highly selective Hox target gene regulation (Sorge, 2012).

Transcriptional regulation in many cases relies on the assembly of regulatory protein complexes mediated by closely spaced TF binding sites within a cis-regulatory module and previous studies have shown that Hox proteins employ this mechanism to control target gene activity in small subsets of cells. The novel HREs were systematically scanned for TF binding motifs appearing in close proximity to Dfd binding sites. Using a statistical test for pair-wise distance distributions, w11 overrepresented DNA motifs for known TFs were found adjoining to Dfd binding sites with 5 of the motifs occurring in both the ChIP-seq and in silico-identified Dfd-HREs. When the expression patterns of six of these transcriptional regulators known to bind to the 11 motifs that were identified were examined, colocalization with Dfd was found in different sub-populations of cells in all cases. Colocalization was already known for two TFs, whose binding sites were coupled to Dfd motifs, including Extradenticle (Exd) , which is known to cooperatively bind with Hox proteins to DNA and thereby increase Hox DNA-binding selectivity. It was next asked whether the short-distance arrangements in Dfd-HREs are of biological relevance and translated into the regulation of similar classes of target genes. To this end, the overrepresentation was statistically tested of expression and biological terms of genes associated with HREs harbouring specific combinations of Dfd and close-by motifs. This analysis revealed that only those Dfd-HREs with short distance intervals between the Dfd and adjacent motifs were coupled to similar gene classes, while random distance intervals did not show any correlation. Strikingly, genes associated with specific short-distance HREs had similar expression and functional annotations as the TFs interacting with the Hox adjoining motifs, suggesting that time and place of Hox action is dictated by spatio-temporally restricted co-regulators. Support for this hypothesis stems from the observation that one of the close-distance partners, Optix, regulates similar processes as Dfd, since Dfd and Optix mutants displayed comparable morphological defects in the head region, such as the absence of mouth hooks, a maxillary segment-derived structure known to be specified by Dfd. In addition, one of the genes associated with a Dfd-Optix HRE, the known Dfd target gene reaper (rpr), is expressed in the ventral epidermis primordium as predicted by its HRE architecture, and regulated by Dfd and Optix in ventral-maxillary cells, which also express these factors. A cell-culture assay using the well-established Dfd responsive module responsible for rpr expression in a few anterior-maxillary cells, the rpr-4S3 Dfd-HRE, with wild-type or mutated Dfd binding sites or reduction of Dfd levels by RNAi confirmed the requirement for simultaneous activity of Dfd and Optix on the rpr-4S3 Dfd-HRE for strong reporter gene induction. Optix binding to the rpr-4S3 Dfd-HRE was additionally confirmed by electrophoretic mobility shift assay (EMSA) experiments. Furthermore, transgenic reporter expression induced by the rpr-4S3 Dfd-HRE was lost in Optix mutant embryos or when the Optix binding sites were mutated. These results demonstrate that Optix, one of the newly identified factors, is a Dfd co-regulator required for proper regulation of the important Hox target gene rpr (Sorge, 2012).

Whether The precise spacing between Hox and adjacent binding sites plays a role for enhancer activity was explored. The rpr-4S3 Dfd HRE, which induces gene expression in a few anterior-maxillary cells, has previously been shown to be under the control of Dfd and Glial cells missing (Gcm), a Dfd co-regulator also identified in this study. Dfd and Gcm as well as Optix binding sites within the rpr-4S3 HRE are directly adjacent to each other, thus a 5- and 10-bp spacer was introduced to interfere with potential interactions of the proteins on the enhancer. In all cases, reporter gene expression was strongly reduced or completely abolished, showing that the close-distance arrangements between Dfd and Gcm as well as Dfd and Optix are required for the in vivo activity of the rpr-4S3 enhancer (Sorge, 2012).

While the results regarding the close-distance arrangement of Dfd and Gcm binding sites suggested the formation of a Dfd-Gcm protein complex, like in the case of Dfd and Exd, only independent binding of the two proteins to the rpr-4S3 enhancer was observed in EMSA experiments , supporting the idea of Hox proteins collaborating with other TFs on target HREs in the absence of physical contact. It has been shown before that Hox proteins together with other TFs that bind in the immediate vicinity recruit non-DNA binding cofactors to HREs. To test if such factors could interact with Dfd and the newly identified short distance binding TFs, the modENCODE data set was scanned and it was found that dCBP/Nej, a member of the CBP/p300 family of transcriptional co-activators bearing acetyltransferase activity, binds to the rpr-4S3 enhancer in vivo. As nej has been previously reported to genetically interact with Dfd, its function was examined in Dfd/Gcm-mediated transcriptional activation. Both factors, Dfd and Gcm, are required for transcriptional activation, since expression of Gcm in Drosophila D.Mel-2 cells, which have basal levels of Dfd activity, resulted in strong induction of reporter gene expression, while abolishing Dfd binding to the rpr-4S3 HRE by mutating all Dfd binding sites or by reducing Dfd protein levels in D.Mel-2 cells using RNAi, strongly reduced reporter gene expression in the presence of Gcm. Strikingly, Dfd- and Gcm-mediated reporter gene expression was strongly reduced in nej dsRNA-treated cells, whereas inhibition of protein deacetylation by Trichostatin A (TSA0) restored reporter gene expression. Consistently, rpr expression was abolished in nej mutant embryos. These results demonstrate that dCBP/Nej-mediated protein acetylation/histone modification is important for the combined activity of Dfd and Gcm on the rpr-4S3 HRE. While it was not possible to demonstrate that nej physically interacts with Dfd protein using various assays, EMSA experiments show that nej interacts with Gcm. Furthermore, acetylation of transiently transfected Gcm was detected in cultured Drosophila cells. Acetylation of Gcm is dependent on Nej, as it was reduced upon RNAi-mediated downregulation of nej. These results are consistent with published work demonstrating that in human cells CBP interacts with Gcma, resulting in its acetylation and stimulation of its transcriptional activity. Since about 10% of all Dfd and nej in vivo genomic binding events during embryonic stages 10-12 overlap, the functional interaction of Dfd and nej observed at the rpr locus does not seem an exception. This finding suggests that the interaction of co-activators (and co-repressors) with Hox proteins and close distance binding TFs on enhancer modules could be a commonly used mechanism to achieve highly specific spatio-temporal control of target gene activity. In this scenario, Hox proteins would control downstream genes by direct transcriptional and/or epigenetic regulation depending on HRE composition and thus cofactor identity and recruitment (Sorge, 2012)

Despite very similar DNA binding behaviour in vitro, Hox proteins regulate distinct morphological features along the anterior-posterior body axis in animal systems. To elucidate the mechanistic basis for the differences in their regulatory properties, Dfd-HREs identified in this study were compared to genomic regions bound by the Hox TF Ultrabithorax (Ubx) at identical developmental stages, as identified by the modENCODE consortium. Searching for overrepresented DNA motifs in both enriched ChIP regions, it was found that Dfd and Ubx bind to identical DNA sequences in vivo, reminiscent to in vitro systems. However, individual binding motifs seem to play only a minor role for Hox binding site selection in vivo, since this analysis revealed that Dfd and Ubx exclusively interact with non-overlapping genomic regions in embryonic stages 9-12. Consequently, Dfd- and Ubx-HREs were found to be associated with distinct classes of genes, revealing that genes with roles in the epidermis are primarily under the control of Dfd at the analysed embryonic stages while genes with mesoderm-related functions are predominantly regulated by Ubx. Consistently, it was found that the expression of tartan (trn), one of the genes associated with a Dfd-HRE, is regulated exclusively by Dfd, but not by Ubx, in epidermal cells, while parcas (pcs), one of the genes linked to a Ubx-HRE is under the selective control of Ubx in mesodermal cells. Furthermore, only Ubx-HREs were found to substantially overlap with cis-regulatory elements stage specifically bound by the mesoderm-specifying TFs Myocyte enhancer factor 2 (Mef2), Twist and Tinman. In contrast, the common ability of both Dfd and Ubx to regulate genes involved in nervous system development was underlined by comparable representations of binding motifs for the neuronal-specifying TFs Asense, Deadpan and Snail in Dfd- and Ubx-HREs (Sorge, 2012).

Strikingly, the basic design principles of Dfd- and Ubx-HREs were found to be similar: like in Dfd-HREs, six binding motifs for known TFs were located adjacent to Ubx binding sites and colocalization studies showed that they are expressed in subsets of Ubx-positive cells. Again, Ubx binding sites and motifs for potential co-regulators occurred most frequently in specific short intervals and only those Ubx-HREs with the preferred distance were associated with specific gene classes. This analysis also revealed that four of the six short-distance motifs were specific for Ubx-HREs, which is consistent with the data showing that Hox proteins interact with different and spatially restricted co-regulators to control target gene expression in selected cells. Importantly, in the cases of the close-distance motifs detected in both HREs, namely the binding sites for the TFs Ladybird early (Lbe) and Cut (Ct), the associated target genes were also expressed in non-overlapping tissues. This raised the question of how different Hox proteins can act on distinct target genes, even when their target HREs exhibit similar binding site compositions including short-distance arrangements. Since Lbe is active in both mesodermal and epidermal cells, one Dfd-Lbe and one Ubx-Lbe HRE was exemplarily analysed, and binding of Lbe protein was confirmed to both HREs by EMSAs. As predicted by the presence of Lbe binding sequences. Complex formation between the Hox protein and Lbe was observed in the case of Ubx and Lbe while Dfd and Lbe interact independently with the Dfd-Lbe HRE, indicating that the two Hox proteins employ different mechanisms for binding to the selected HREs. Lbe interaction with the Dfd-Lbe and Ubx-Lbe HREs is essential for in vivo activity, since in both cases ectopic reporter gene expression was observed when Lbe binding sites were mutated. Even more important, reporter gene expression was specifically changed only in segments in which either Dfd or Ubx is active, meaning in the case of the Dfd-Lbe HRE in maxillary cells and in the case of the Ubx-Lbe HRE in abdominal segments A1-A7. Taken together, these results demonstrate that the combined activity of Lbe and the Hox proteins Dfd or Ubx on selected HREs is critical for the precise spatiotemporal and segment-specific control of HRE activity. It was next asked whether additional (DNA- and non-DNA-binding) factors contribute to the predicted cell type-specific expression of the Dfd-Lbe and Ubx-Lbe HREs. Using the Drosophila Interactions Database (DroID; Murali, 2011) and published genome-wide DNA binding studies a search was carried out for unique Dfd-lbe and Ubx-lbe interactors. It was discovered that almost 20% of all Ubx-Lbe HREs but none of the Dfd-Lbe HREs were found to interact with the mesoderm-specifying factor Mef2 in vivo, while H3K9me3 histone marks, which are mediated by one of the unique Dfd-lbe interactors, Enhancer of zeste E(z), are enriched only within Dfd-Lbe HREs. Interestingly, E(z) modifies chromatin also by trimethylating H3K27 residues, a histone mark highly enriched at the genomic region spanning the ChIP-detected Dfd-Lbe HRE. Consistent with the repressive function of this histone modification, loss of Lbe binding to the Dfd-Lbe HRE results in ectopic reporter gene expression, suggesting that Lbe (and Dfd) recruits E(z) to the Dfd-Lbe HRE for cell type-specific target gene repression (Sorge, 2012).

Taken together, these results demonstrate that Hox proteins interact with different regulatory proteins on HREs, which allows them to differentially regulate their target genes despite their similar DNA binding properties. The fact that these interactions occur only in a few cells for a short period of time is very likely one of the major reasons why the identification of factors conferring regulatory precision and specificity to Hox function has met with little success so far (Sorge, 2012).

This study, has identified crucial features of HREs, which are essential for cell type-specific regulation of Hox target genes in vivo. In addition to motif composition the exact spatial arrangement of TF binding elements is critical to translate Dfd function into transcriptional regulation in vivo. These architectural features of Dfd-HREs alone accurately predict target gene function and expression patterns. Furthermore, it was found that epigenetic regulators bind to HREs on a genome-wide scale, suggesting that they generally collaborate with Hox proteins to achieve stable target gene regulation. This is in line with recent findings showing that chromatin modifications at enhancers strongly correlate with functional enhancer activity and tissue specificity. By comparing HREs regulated by Dfd and Ubx, two different Hox proteins with different embryonic regulatory specificities, this study shows that while similar design principles apply, specificity is encoded by distinct sets of co-occurring DNA motifs. Due to the highly dynamic regulatory output of Hox TFs in space and time, cell type-specific approaches are required in future to elucidate all relevant aspects of Hox-chromatin and Hox-cofactor interactions (Sorge, 2012).

A p53 enhancer region regulates target genes through chromatin conformations in cis and in trans

How a p53 enhancer transmits regulatory information was examined in vivo. Using genetic ablation together with digital chromosome conformation capture and fluorescent in situ hybridization, this study found that a Drosophila p53 enhancer region (referred to as the p53 response element [p53RE]) physically contacts targets in cis and across the centromere to control stress-responsive transcription at these sites. Furthermore, when placed at ectopic genomic positions, fragments spanning this element re-established chromatin contacts and partially restore target gene regulation to mutants lacking the native p53RE. Therefore, a defined p53 enhancer region is sufficient for long-range chromatin interactions that enable multigenic regulation (Link, 2013).

This study present in vivo functional evidence that a single enhancer region can specify regulation of multiple targets in cis and in trans. Using tailored deletions, it was found that a p53 regulatory element controlled stimulus-dependent induction of multiple genes, with effects on targets that range from 4 kb to 330 kb throughout the Drosophila Reaper region. In these studies, the p53RE also regulated xrp1, a genetically unlinked target residing across the centromere. Furthermore, when transplanted to ectopic locations, contacts with target sites were re-established and regulation of some target genes was restored. Together, these functional studies offer compelling evidence that an enhancer transmits regulatory activity in trans through direct physical contact (Link, 2013).

In principle, long-range regulation of xrp1 by the native p53RE could involve local induction of an activator that subsequently induces distant genes, but this type of expression cascade would not explain the data. First, no correlation exists between the timing of RIPD gene induction and proximity to the p53RE. Second, cis targets in the Reaper interval encode products with no known function in the nucleus or in transcription. Third, conventional expression cascades would not account for the restoration of regulation and contacts by a transgenic rescue fragment. Therefore, the idea is favored that long-range regulation by the p53RE involves chromosomal architectures that link this enhancer to target genes regardless of whether they are in cis or in trans (Link, 2013).

Using either 3C or direct visualization, suggestive chromatin links between enhancers and distant genomic sites in trans have been reported. Few have been genetically tested, and, where functionally studied, detectable effects were not seen. the current finding that productive looping contacts can be assembled from a foreign site suggests that determinants of long-range chromatin interactions are modular and probably specified through sequence motifs, secondary structures, and epigenetic features that occur in vivo. It is further noted that the presence of contacts is not sufficient for target induction. For example, despite loops between the native p53RE and sites near grim or contacts between the ectopic p53RE and sites near rpr and skl, transcriptional induction was not seen. Therefore, elements that map outside of the rescue fragment or constraints imposed by flanking chromatin may also be important (Link, 2013).

Given that p53 enhancers in both flies and humans share a common sequence motif, mechanisms by which these response elements form long-range interactions in trans may be conserved. It will be interesting to see whether other enhancer regions share this property. Likewise, it will be important to determine whether these contacts are mediated through complexes involving proteins such as Cohesin, Mediator, Ldb1, Polycomb, or CTCF. If broadly generalized, the precedent established here could offer a framework that helps explain genetic disease alleles mapping to noncoding sequences (Link, 2013).

The bHLH-PAS transcription factor Dysfusion regulates tarsal joint formation in response to Notch activity during Drosophila leg development

A characteristic of all arthropods is the presence of flexible structures called joints that connect all leg segments. Drosophila legs include two types of joints: the proximal or 'true' joints that are motile due to the presence of muscle attachment and the distal joints that lack musculature. These joints are not only morphologically, functionally and evolutionarily different, but also the morphogenetic program that forms them is distinct. Development of both proximal and distal joints requires Notch activity; however, it is still unknown how this pathway can control the development of such homologous although distinct structures. This study shows that the bHLH-PAS transcription factor encoded by the gene dysfusion (dys), is expressed and absolutely required for tarsal joint development while it is dispensable for proximal joints. In the presumptive tarsal joints, Dys regulates the expression of the pro-apoptotic genes and head involution defective and the expression of the RhoGTPases modulators, RhoGEf2 and RhoGap71E, thus directing key morphogenetic events required for tarsal joint development. When ectopically expressed, dys is able to induce some aspects of the morphogenetic program necessary for distal joint development such as fold formation and programmed cell death. This novel Dys function depends on its obligated partner Tango to activate the transcription of target genes. A dedicated dys cis-regulatory module was identified that regulates dys expression in the tarsal presumptive leg joints through direct Su(H) binding. All these data place dys as a key player downstream of Notch, directing distal versus proximal joint morphogenesis (Cordoba, 2014: PubMed).

Targets of Activity

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).

Domain structure of Reaper and Reaper's killing activity

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).

Mitochondrial localization of Reaper to promote inhibitors of apoptosis protein degradation conferred by GH3 domain-lipid interactions

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).

Antisense-mediated depletion reveals essential and specific functions of microRNAs in Drosophila development: Differential posttranscriptional repression of the proapoptotic factors hid, grim, reaper, and sickle

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).

reaper : Biological Overview | Evolutionary Homologs | Protein Interactions and parallel pathways | Developmental Biology | Effects of Mutation | References

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