InteractiveFly: GeneBrief

Ecdysone-induced protein 93F: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Steroid hormones coordinate multiple cellular changes, yet the mechanisms by which these systemic signals are refined into stage- and tissue-specific responses remain poorly understood. The Drosophila gene Eip93F, more familiarly termed E93 determines the nature of a steroid-induced biological response. E93 mutants possess larval salivary glands that fail to undergo steroid-triggered programmed cell death, and E93 is expressed in cells immediately before the onset of death. E93 protein is bound to the sites of steroid-regulated and cell death genes on polytene chromosomes, and the expression of these genes is defective in E93 mutants. Furthermore, expression of E93 is sufficient to induce programmed cell death. It is proposed that the steroid induction of E93 determines a programmed cell death response during development (Lee, 2000).

The mechanisms of steroid signaling have been extensively studied in Drosophila larval salivary glands by virtue of the giant polytene chromosomes that form ecdysone-induced puffs, reflecting a transcriptional regulatory hierarchy. The ecdysone receptor complex, a heterodimer of the EcR and Usp nuclear receptors, activates transcription of a small set of early regulatory genes. These genes encode transcription factors that, in turn, activate a larger set of late genes, which are thought to play a more direct role in controlling the appropriate biological responses to the hormone. Previous studies have implicated the EcR, usp, βFTZ-F1, BR-C, and E74A genes in steroid-activated larval cell death. The role of βFTZ-F1 appears to be indirect, functioning as general competence factor for prepupal responses to ecdysone. In contrast, EcR, Usp, BR-C, and E74A play a more direct role in triggering salivary gland cell death through the coordinate induction of rpr and hid transcription. These factors, however, are not sufficient for the death response because they do not direct this pathway in response to the earlier pulse of ecdysone at puparium formation. Rather, one or more stage-specific regulators must be induced by ecdysone at the end of prepupal development that determine the stage specificity of salivary gland cell death. The E93 early gene is an ideal candidate for fulfilling this function. E93 is induced as a primary reponse to ecdysone in a stage- and tissue-specific manner (Baehrecke, 1995). E93 transcription increases immediately prior to larval midgut and salivary gland cell death and is coordinately induced with rpr and hid (Baehrecke, 1995; Jiang, 1997). This correlation suggests that E93 may contribute to the stage specificity of larval tissue cell death (Lee, 2000).

The expression of E93 protein in dying cells, combined with the defects in E93 mutant salivary gland cell death and transcription of apoptosis genes, indicates that E93 is a key determinant of steroid-induced programmed cell death. Therefore, tests were performed to see if expression of E93 is sufficient to kill wing imaginal disc cells that have a well-defined response to ecdysone during metamorphosis. This experiment was carried out by crossing UAS-E93 transformant flies with Drosophila strains that express GAL4 in wing imaginal discs (vg-GAL4). All progeny that possess both UAS-E93 and vg-GAL4 die at the start of pupal development. This lethal phase, combined with the wealth of information about ecdysone-triggered wing development led to a detailed characterization of E93-induced cell death in vg-GAL4 UAS-E93 animals. Control (UAS-E93 alone or vg-GAL4 alone) wing-thoracic imaginal discs dissected 2 hr following puparium formation exhibit little cell death. By contrast, E93-expressing wing-thoracic imaginal discs, exhibit extensive cell death in the wing blade and hinge regions at this developmental stage. This pattern of cell death mimics the pattern of GAL4 expression in this vg-GAL4 strain of Drosophila, as determined by crossing vg-GAL4 flies with a UAS-lacZ reporter and detecting β-galactosidase activity. Animals expressing E93 under the control of vg-GAL4 die soon after head eversion during metamorphosis and exhibit defects in the presumptive adult notum and wing. These animals were examined further by characterizing the morphology of wing imaginal discs prior to the lethal phase but after the induction of ectopic cell death. While control wing-thoracic imaginal discs dissected from animals 4 hr following puparium formation have clearly progressed in elongation of the wing, E93-expressing animals of the same age possess defective wing-thoracic imaginal discs that do not properly elongate. These data demonstrate that expression of E93 is sufficient to induce programmed cell death (Lee, 2000).

It is concluded that the precise temporal and spatial patterns of E93 induction by the steroid hormone ecdysone determines the biological fate of those target tissues, directing the massive programmed cell death of the larval salivary glands during metamorphosis. E93 also acts both directly and indirectly to regulate the transcription of key effector genes that drive the cell death response. Initial studies of the ecdysone-triggered gene cascades speculated that early ecdysone-induced regulatory genes might be expressed in a tissue-specific manner, directing the different fates of larval and adult cells during metamorphosis. In contrast, molecular characterization of the BR-C, E74, and E75 early genes demonstrates that these genes are widely expressed throughout the animal. Localization of EcR and BR-C protein isoforms reveals that they are expressed in subsets of ecdysone target tissues; however, these expression patterns do not correlate with sets of tissues that undergo one fate in response to ecdysone. Similarly, studies of EcR, usp, BR-C, and E74 mutants have revealed multiple functions for these genes, affecting the development of both larval and adult cells during metamorphosis. These observations led to the 'tissue coordination model', which proposes that overlapping combinations of early ecdysone-induced transcription factors dictate the proper tissue-specific responses to ecdysone pulses during development (Lee, 2000).

E93 stands in sharp contrast to these widely expressed early genes. E93 expression is restricted to metamorphosis, with induction in the midguts of newly formed prepupae preceding induction in the salivary glands of late prepupae. The temporal correlation of this expression pattern with the onset of midgut and salivary gland cell death raised the possibility that E93 might play a role in regulating the death response (Baehrecke, 1995). Strong evidence in support of this model is proved. Antibody stains show that E93 is induced in a cell type-specific pattern in the larval midguts, restricted to the polytene larval cells that are fated to die, and excluded from the diploid imaginal cells that form the adult gut. Similarly, E93 protein expression in the salivary gland parallels that of its mRNA, immediately preceding cell death. E93 mutants die as pupae with persistent salivary glands, and this salivary gland cell death defect can be rescued by E93 expression from a transgene. Moreover, ectopic E93 expression is sufficient to direct a death response. Thus, the ecdysone induction of E93 defines the fate of that tissue, directing its immediate and massive destruction through programmed cell death. E93 regulation therefore provides a molecular mechanism for refining the systemic ecdysone signal into a specific biological response during development (Lee, 2000).

Although E93 encodes a novel protein with little similarity to other proteins in the sequence databases, it shares several characteristics of Drosophila transcription factors. These include homopolymeric tracts of acidic amino acids that can serve as transcriptional activation domains, a potential nuclear localization signal, and two basic domains that could serve as DNA binding motifs. E93 is localized to nuclei and binds to specific sites on polytene chromosomes, further suggesting that E93 regulates gene activity. Significantly E93 mutants impact the transcription of genes from chromosome loci that are bound by E93 protein. While these data do not provide conclusive evidence that distinguish the biochemical characteristics of DNA and chromatin binding, these results are consistent with the hypothesis that E93 encodes a novel transcription regulator (Lee, 2000).

E93 appears to exert its effects by both directly and indirectly regulating genes required for programmed cell death. Prior to the prepupal stage, ecdysone triggers regulatory hierarchies that do not result in salivary gland cell death. In late third instar larvae, for example, the ecdysone receptor complex activates the primary response genes BR-C, E74, and E75. These early genes, in turn, direct a switch in salivary gland late gene expression, repressing the glue genes and inducing more than 100 late genes including the L71 genes. The following pulse of ecdysone, at the end of prepupal development, triggers the BR-C, E74, E75, and E93 early genes. This response is dependent on the prior expression of the βFTZ-F1 competence factor, which is necessary and sufficient for early gene induction in late prepupae. As expected βFTZ-F1 is expressed at a normal level in E93 mutants with a delay due to genetic background. In contrast E93 is required for ecdysone induction of the BR-C, E74A, and E75A genes in prepupal salivary glands. E93 mutants do not impact EcR and E74B transcription and head eversion, indicating that the prepupal pulse of ecdysone is normal in these animals. Thus, the effect of E93 mutants on transcription of early genes is not caused by the absence of ecdysone. E93 protein binds to the 74EF and 75B puffs that contain the E74 and E75 genes, suggesting that these are direct regulatory targets. E93 does not bind to the 2B5 puff containing the BR-C gene, suggesting that this regulation is indirect (Lee, 2000).

Several cell death genes are transcribed immediately prior to larval salivary gland programmed cell death. Components of the core apoptosis machinery, including Ark and the caspase dronc, as well as the death genes rpr, hid, and crq, increase in transcription in late prepupal salivary glands. The synchronous induction of these cell death genes indicates that salivary glands die by a mechanism that is similar to that utilized in apoptosis during Drosophila embryogenesis, where rpr and hid are involved in caspase activation. The increase in crq transcription in dying salivary glands suggests that these cells are unique, however, since Crq is expressed in phagocytes and functions in removal of dying cells during embryogenesis. This increase in crq transcription is not due to the adhesion of phagocytes to the dying salivary gland, since Crq protein is expressed at a high level in the dying cells. While salivary gland cell destruction involves genes that function in apoptosis, these cells also have characteristics of autophagy, and this form of cell death may utilize crq in the terminal stages of cell removal (Lee, 2000).

E93 mutants impact transcription of cell death genes, consistent with the model that E93 serves as a regulator that specifies the cell death response to ecdysone. E93 mutants exhibit defects in transcription of rpr, hid, crq, ark, and dronc. The 75C locus that contains rpr and hid and the 53F locus that contains ark are not bound by E93 protein, suggesting that the regulation of these genes is indirect. Recent studies of rpr and hid regulation support this conclusion, since mutations in BR-C and E74A alter rpr and hid RNA levels, and E93 is required for BR-C and E74A expression. The 21C locus, which contains crq, is bound by E93 protein, suggesting that E93 may directly regulate crq transcription. Thus, E93 plays an essential role in regulating cell death genes, thereby directing steroid-triggered programmed cell death (Lee, 2000).

This study demonstrates that components of the central cell death pathway, including Ark and dronc, exhibit dynamic changes in RNA transcription that immediately precede salivary gland cell death. It is important to consider that many of these factors may also be regulated at the posttranscriptional level. For example, rpr and hid direct programmed cell death during Drosophila embryogenesis by repressing the inhibitory activity of DIAP1 on caspase activation. Thus, while rpr, hid, and dronc are regulated by the ecdysone-induced primary response genes at the transcriptional level, dronc may also be regulated by a secondary mechanism. Future studies of the genetic pathways that mediate steroid-regulated destruction of larval salivary glands will provide further insights into the conserved molecular mechanisms that underlie cell death during development (Lee, 2000).

Steroid hormone induction of temporal gene expression in Drosophila brain neuroblasts generates neuronal and glial diversity

An important question in neuroscience is how stem cells generate neuronal diversity. During Drosophila embryonic development, neural stem cells (neuroblasts) sequentially express transcription factors that generate neuronal diversity; regulation of the embryonic temporal transcription factor cascade is lineage-intrinsic. In contrast, larval neuroblasts generate longer ~50 division lineages, and currently only one mid-larval molecular transition is known: Chinmo/Imp/Lin-28+ neuroblasts transition to Syncrip+ neuroblasts. This study shows that the hormone ecdysone is required to down-regulate Chinmo/Imp and activate Syncrip, plus two late neuroblast factors, Broad and E93. Seven-up triggers Chinmo/Imp to Syncrip/Broad/E93 transition by inducing expression of the Ecdysone receptor in mid-larval neuroblasts, rendering them competent to respond to the systemic hormone ecdysone. Importantly, late temporal gene expression is essential for proper neuronal and glial cell type specification. This is the first example of hormonal regulation of temporal factor expression in Drosophila embryonic or larval neural progenitors (Syed, 2017).

This study shows that the steroid hormone ecdysone is required to trigger a major gene expression transition at mid-larval stages: central brain neuroblasts transition from Chinmo/Imp to Broad/Syncrip/E93. Furthermore, it was shown that Svp activates expression of EcR-B1 in larval neuroblasts, which gives them competence to respond to ecdysone signaling, thereby triggering this gene expression transition. Although a global reduction of ecdysone levels is likely to have pleiotropic effects on larval development, multiple experiments were performed to show that the absence or delay in late temporal factor expression following reduced ecdysone signaling is not due to general developmental delay. First, the EcR gene itself is expressed at the normal time (~56 hr) in the whole organism ecdysoneless¹ mutant, arguing strongly against a general developmental delay. Second, a type II neuroblast seven-up mutant clone shows a complete failure to express EcR and other late factors, in the background of an entirely wild type larvae; this is perhaps the strongest evidence that the phenotypes that are described are not due to a general developmental delay. Third, lineage-specific expression of EcR dominant negative leads to loss of Syncrip and E93 expression without affecting Broad expression; the normal Broad expression argues against a general developmental delay. Fourth, live imaging was used to directly measure cell cycle times, and it was found that lack of ecdysone did not slow neuroblast cell cycle times. Taken together, these data support the conclusion that ecdysone signaling acts directly on larval neuroblasts to promote an early-to-late gene expression transition (Syed, 2017).

The role of ecdysone in regulating developmental transitions during larval stages has been well studied; it can induce activation or repression of suites of genes in a concentration dependent manner. Ecdysone induces these changes through a heteromeric complex of EcR and the retinoid X receptor homolog Ultraspiracle. Ecdysone is required for termination of neuroblast proliferation at the larval/pupal transition, and is known to play a significant role in remodeling of mushroom body neurons and at neuromuscular junctions. This study adds to this list another function: to trigger a major gene expression transition in mid-larval brain neuroblasts (Syed, 2017).

Does ecdysone signaling provide an extrinsic cue that synchronizes larval neuroblast gene expression? Good coordination of late gene expression is not seen, arguing against synchronization. For example, Syncrip can be detected in many neuroblasts by 60 hr, whereas Broad appears slightly later at ~72 hr, and E93 is only detected much later at ~96 hr, by which time Broad is low. This staggered expression of ecdysone target genes is reminiscent of early and late ecdysone-inducible genes in other tissues. In addition, for any particular temporal factor there are always some neuroblasts expressing it prior to others, but not in an obvious pattern. It seems the exact time of expression can vary between neuroblasts. Whether the pattern of response is due to different neuroblast identities, or a stochastic process, remains to be determined (Syed, 2017).

It has been shown preiously that the Hunchback-Krüppel-Pdm-Castor temporal gene transitions within embryonic neuroblasts are regulated by neuroblast-intrinsic mechanisms: they can occur normally in neuroblasts isolated in culture, and the last three factors are sequentially expressed in G2-arrested neuroblasts. Similarly, optic lobe neuroblasts are likely to undergo neuroblast-intrinsic temporal transcription factor transitions, based on the observation that these neuroblasts form over many hours of development and undergo their temporal transitions asynchronously. In contrast, this study shows that ecdysone signaling triggers a mid-larval transition in gene expression in all central brain neuroblasts (both type I and type II). Although ecdysone is present at all larval stages, it triggers central brain gene expression changes only following Svp-dependent expression of EcR-B1 in neuroblasts. Interestingly, precocious expression of EcR-B1 (worniu-gal4 UAS-EcR-B1) did not result in premature activation of the late factor Broad, despite the forced expression of high EcR-B1 levels in young neuroblasts. Perhaps there is another required factor that is also temporally expressed at 56 hr. It is also noted that reduced ecdysone signaling in ecd^ts mutants or following EcRDN expression does not permanently block the Chinmo/Imp to Broad/Syncrip/E93 transition; it occurs with variable expressivity at 120-160 hr animals (pupariation is significantly delayed in these ecd^ts mutants), either due to a failure to completely eliminate ecdysone signaling or the presence of an ecdysone-independent mechanism (Syed, 2017).

A small but reproducible difference was found in the effect of reducing ecdysone levels using the biosynthetic pathway mutant ecd^ts versus expressing a dominant negative EcR in type II neuroblasts. The former genotype shows a highly penetrant failure to activate Broad in old neuroblasts, whereas the latter genotype has normal expression of Broad (despite failure to down-regulate Chinmo/Imp or activate E93). This may be due to failure of the dominant negative protein to properly repress the Broad gene. Differences between EcRDN and other methods of reducing ecdysone signaling have been noted before (Syed, 2017).

Drosophila Svp is an orphan nuclear hormone receptor with an evolutionarily conserved role in promoting a switch between temporal identity factors. In Drosophila, Svp it is required to switch off hunchback expression in embryonic neuroblasts, and in mammals the related COUP-TF1/2 factors are required to terminate early-born cortical neuron production, as well as for the neurogenic to gliogenic switch. This study showed that Svp is required for activating expression of EcR, which drives the mid-larval switch in gene expression from Chinmo/Imp to Syncrip/Broad/E93 in central brain neuroblasts. The results are supported by independent findings that svp mutant clones lack expression of Syncrip and Broad in old type II neuroblasts (Tsumin Lee, personal communication to Chris Doe). Interestingly, Svp is required for neuroblast cell cycle exit at pupal stages, but how the early larval expression of Svp leads to pupal cell cycle exit was a mystery. The current results provide a satisfying link between these findings: Svp was shown to activate expression of EcR-B1, which is required for the expression of multiple late temporal factors in larval neuroblasts. Any one of these factors could terminate neuroblast proliferation at pupal stages, thereby explaining how an early larval factor (Svp) can induce cell cycle exit five days later in pupae. It is interesting that one orphan nuclear hormone receptor (Svp) activates expression of a second nuclear hormone receptor (EcR) in neuroblasts. This motif of nuclear hormone receptors regulating each other is widely used in Drosophila, C. elegans, and vertebrates (Syed, 2017).

The position of the Svp+ neuroblasts varied among the type II neuroblast population from brain-to-brain, suggesting that Svp may be expressed in all type II neuroblasts but in a transient, asynchronous manner. This conclusion is supported by two findings: the svp-lacZ transgene, which encodes a long-lived β-galactosidase protein, can be detected in nearly all type II neuroblasts; and the finding that Svp is required for EcR expression in all type II neuroblasts, consistent with transient Svp expression in all type II neuroblasts. It is unknown what activates Svp in type II neuroblasts; its asynchronous expression is more consistent with a neuroblast-intrinsic cue, perhaps linked to the time of quiescent neuroblast re-activation, than with a lineage-extrinsic cue. It would be interesting to test whether Svp expression in type II neuroblasts can occur normally in isolated neuroblasts cultured in vitro, similar to the embryonic temporal transcription factor cascade (Syed, 2017).

Castor and its vertebrate homolog Cas-Z1 specify temporal identity in Drosophila embryonic neuroblast lineages and vertebrate retinal progenitor lineages, respectively (Mattar, 2015). Although this study shows that Cas is not required for the Chinmo/Imp to Syncrip/Broad/E93 transition, it has other functions. Cas expression in larval neuroblasts is required to establish a temporal Hedgehog gradient that ultimately triggers neuroblast cell cycle exit at pupal stages (Syed, 2017).

Drosophila embryonic neuroblasts change gene expression rapidly, often producing just one progeny in each temporal transcription factor window. In contrast, larval neuroblasts divide ~50 times over their 120 hr lineage. Mushroom body neuroblasts make just four different neuronal classes over time, whereas the AD (ALad1) neuroblast makes ~40 distinct projection neuron subtypes. These neuroblasts probably represent the extremes (one low diversity, suitable for producing Kenyon cells; one high diversity, suitable for generating distinct olfactory projection neurons). This study found that larval type II neuroblasts undergo at least seven molecularly distinct temporal windows. If it is assumed that the graded expression of Imp (high early) and Syncrip (high late) can specify fates in a concentration-dependent manner, many more temporal windows could exist (Syed, 2017).

This study illuminates how the major mid-larval gene expression transition from Chinmo/Imp to Broad/Syncrip/E93 is regulated; yet many new questions have been generated. What activates Svp expression in early larval neuroblasts - intrinsic or extrinsic factors? How do type II neuroblast temporal factors act together with Dichaete, Grainy head, and Eyeless INP temporal factors to specify neuronal identity? Do neuroblast or INP temporal factors activate the expression of a tier of 'morphogenesis transcription factors' similar to leg motor neuron lineages? What are the targets of each temporal factor described here? What types of neurons (or glia) are made during each of the seven distinct temporal factor windows, and are these neurons specified by the factors present at their birth? The identification of new candidate temporal factors in central brain neuroblasts opens up the door for addressing these and other open questions (Syed, 2017).

Hormone-dependent control of developmental timing through regulation of chromatin accessibility

Specification of tissue identity during development requires precise coordination of gene expression in both space and time. Spatially, master regulatory transcription factors are required to control tissue-specific gene expression programs. However, the mechanisms controlling how tissue-specific gene expression changes over time are less well understood. This study shows that hormone-induced transcription factors control temporal gene expression by regulating the accessibility of DNA regulatory elements. Using the Drosophila wing, it was demonstrated that temporal changes in gene expression are accompanied by genome-wide changes in chromatin accessibility at temporal-specific enhancers. A temporal cascade of transcription factors was uncovered following a pulse of the steroid hormone ecdysone such that different times in wing development can be defined by distinct combinations of hormone-induced transcription factors. Finally, the ecdysone-induced transcription factor E93 was shown to control temporal identity by directly regulating chromatin accessibility across the genome. Notably, it was found that E93 controls enhancer activity through three different modalities, including promoting accessibility of late-acting enhancers and decreasing accessibility of early-acting enhancers. Together, this work supports a model in which an extrinsic signal triggers an intrinsic transcription factor cascade that drives development forward in time through regulation of chromatin accessibility (Uyehara, 2017).

The importance of master transcription factors in specifying spatial identity during development suggests that they may control where other transcription factors bind in the genome. One prediction of this model is that tissues whose identities are determined by different master transcription factors would exhibit different genome-wide DNA-binding profiles. However, it was recently found that the Drosophila appendages (wings, legs, and halteres), which use different transcription factors to determine their identities, share nearly identical open chromatin profiles. Moreover, these shared open chromatin profiles change coordinately over developmental time. There are two possible explanations for these findings. Either (1) different transcription factors produce the same open chromatin profiles in different appendages or (2) transcription factors shared by each appendage control open chromatin profiles instead of the master transcription factors of appendage identity. The second model is favored for several reasons. Since the appendage master transcription factors possess different DNA-binding domains with distinct DNA-binding specificities, it is unlikely for them to bind the same sites in the genome. Supporting this expectation, ChIP for Scalloped and Homothorax, two transcription factors important for appendage identity, shows clear tissue-specific binding in both the wing and eye–antennal imaginal discs. The second model is also preferred because it provides a relatively straightforward mechanism for the observed temporal changes in open chromatin: By changing the expression of the shared temporal transcription factor over time, the open chromatin profiles that it controls would change as well. In contrast, expression of appendage master transcription factors is relatively stable over time, making it unlikely for them to be sufficient for temporal changes in open chromatin (Uyehara, 2017).

It is proposed that control of chromatin accessibility in the appendages is mediated at least in part by transcription factors downstream from ecdysone signaling. According to this model, a systemic pulse of ecdysone initiates a temporal cascade of hormone-induced transcription factor expression in each of the appendages. These are referred to as 'temporal' transcription factors. Temporal transcription factors can directly regulate the accessibility of transcriptional enhancers by opening or closing them, thereby conferring temporal specificity to their activity and driving development forward in time. Master transcription factors then bind accessible enhancers depending on their DNA-binding preferences (or other means of binding DNA) and differentially regulate the activity of these enhancers to control spatial patterns of gene expression, thus shaping the unique identities of individual appendages (Uyehara, 2017).

The experiments with E93 provide direct support for this model. In wild-type wings, thousands of changes in open chromatin occur after the large pulse of ecdysone that triggers the end of larval development. In E93 mutants, ~40% of these open chromatin changes fail to occur. Importantly, nearly three-quarters of sites that depend on E93 for accessibility correspond to temporally dynamic sites in wild-type wings. Thus, chromatin accessibility is not grossly defective across the genome; instead, defects occur specifically in sites that change in accessibility over time. This finding, combined with the large fraction of temporally dynamic sites that depend on E93 for accessibility, indicates that E93 controls a genome-wide shift in the availability of temporal-specific transcriptional enhancers. Supporting this hypothesis, temporal-specific enhancers depend on E93 for both accessibility and activity. Since it is proposed that the response to ecdysone is shared across the appendages, it is predicted that similar defects occur in appendages besides the wing. It remains to be seen whether other ecdysone-induced transcription factors besides E93 control accessibility of enhancers at different developmental times. It also remains to be seen how the temporal transcription factors work with the appendage master transcription factors to control appendage-specific enhancer activity (Uyehara, 2017).

The findings suggest that E93 controls temporal-specific gene expression through three different modalities that potentially rely on three distinct biochemical activities. The enrichment of E93 motifs and binding of E93 to temporally dynamic sites indicate that it contributes to this regulation directly. It is proposed that these combined activities drive development forward in time by turning off early-acting enhancers and simultaneously turning on late-acting enhancers (Uyehara, 2017).

First, as in the case of the tenectin tnc^blade enhancer, active most strongly in the interveins between the first and second and between the fourth and fifth longitudinal veins and in cells near the proximal posterior margin, E93 appears to function as a conventional activator. In the absence of E93, tnc^blade fails to express at high levels, but the accessibility of the enhancer does not measurably change. This suggests that binding of E93 to tnc^blade is required to recruit an essential coactivator. Importantly, this finding demonstrates that E93 is not solely a regulator of chromatin accessibility. E93 binds many open chromatin sites in the genome without regulating their accessibility and thus may regulate the temporal-specific activity of many other enhancers. In addition, since the tnc^blade enhancer opens between L3 and 24 h even in the absence of E93, there must be other factors that control its accessibility, perhaps, for example, transcription factors induced by ecdysone earlier in the temporal cascade (Uyehara, 2017).

Second, as in the case of the nub^vein enhancer, E93 is required to promote chromatin accessibility. In this capacity, E93 may function as a pioneer transcription factor to open previously inaccessible chromatin. Alternatively, E93 may combine with other transcription factors, such as the wing master transcription factors, to compete nucleosomes off DNA. Testing the ability of E93 to bind nucleosomal DNA will help to discriminate between these two alternatives. In either case, it is proposed that this function of E93 is necessary to activate late-acting enhancers across the genome. Since only half of E93-dependent enhancers are directly bound by E93 at 24 h, it is also possible that E93 regulates the expression of other transcription factors that control chromatin accessibility. Alternatively, if E93 uses a “hit and run” mechanism to open these enhancers, the ChIP time point may have been too late to capture E93 binding at these sites (Uyehara, 2017).

Finally, as in the case of the broad br^disc enhancer, E93 is required to decrease chromatin accessibility. It is proposed that this function of E93 is necessary to inactivate early-acting enhancers across the genome. Current models of gene regulation do not adequately explain how sites of open chromatin are rendered inaccessible, but the ability to turn off early-acting enhancers is clearly an important requirement in developmental gene regulation. It may also be an important contributor to diseases such as cancer, which exhibits widespread changes in chromatin accessibility relative to matched normal cells. Thus, this role of E93 may represent a new functional class of transcription factor (“reverse pioneer”) or conventional transcriptional repressor activity. Additional work is required to decipher the underlying mechanisms. Notably, recent work on the temporal dynamics of iPS cell reprogramming suggest a similar role for Oct4, Sox2, and Klf4 in closing open chromatin to inactivate somatic enhancers (Chronis, 2017; Uyehara, 2017 and references therein).

REGULATION

Transcriptional Regulation

Premature expression of the late FTZ-F1 protein has an effect on early gene induction by ecdysone. The inability of E93 to be induced by ecdysone in late-third instar larval salivary glands can be overcome by ectopic expression of FTZ-F1. FTZ-F1 also represses its own transcription (Woodard, 1994).

The steroid hormone ecdysone induces a precise sequence of gene activity in Drosophila melanogaster salivary glands in late third instar larvae. The acquisition of competence for this response does not result from a single event or pathway but requires factors that accumulate throughout the instar. Individual transcripts become competent to respond at different times and their expression is differentially affected in ecd1, dor22 and BR-C mutants. ecd1 mutants are deficient in ecdysone. dor encodes a protein with a zinc-finger like motif. The induction of early-late transcripts, originally assumed to necessarily follow early transcripts, is partially independent of early transcript activation. Attempts to inhibit the synthesis of regulatory proteins reveal transcript-specific superinduction effects. Furthermore these inhibitors led to the induction of betaFTZ-F1 and E93 transcripts at levels normally found in prepupal glands. These studies reveal the complexity of the processes underlying the establishment of a hormonal response (Richards, 1999).

The beta FTZ-F1 orphan nuclear receptor functions as a competence factor for stage-specific responses to the steroid hormone ecdysone during Drosophila metamorphosis. beta FTZ-F1 mutants pupariate normally in response to the late larval pulse of ecdysone but display defects in stage-specific responses, adult head eversion, leg elongation and salivary gland death, in response to the subsequent ecdysone pulse in prepupae. The ecdysone-triggered genetic hierarchy that directs these developmental responses is severely attenuated in beta FTZ-F1 mutants, although ecdysone receptor expression is unaffected. Both E74A and E75A, whose levels of expression are normally increased several orders of magnitude by ecdysone, are significantly affected in betaFTZ-F1 mutants. The severity of these effects correlates with the intensity of polytene chromosome staining by FTZ-F1 antibodies. The Br-C locus is only weakly stained, while E74 is strongly stained, and E75 is the most intensely stained site in the genome. It thus appears that betaFTZ-F1 exerts specificity to the degree to which it can enhance the ecdysone-induction of different promoters. The E93 early gene is also submaximally induced in betaFTZ-F1 mutants, consistent with the proposal that this stage-specific response is dependent on betaFTZ-F1 function. In contrast, the levels of Ecdysone receptor and Ultraspiracle mRNA are not significanty affected by betaFTZ-F1. EDG84A, a gene that encodes a pupal cuticle protein that is specifically expressed in the imaginal discs of mid-prepupae, contains a betaFTZ-F1 binding site upstream from the start site, and EDG84A transcription is delayed and reduced in betaFTZ-F1 mutants. Thus this study defines beta FTZ-F1 as an essential competence factor for stage-specific responses to a steroid signal and implicates interplay among nuclear receptors as a mechanism for achieving hormonal competence (Broadus, 1999).

Targets of Activity

The nuclear localization of E93 in larval salivary glands provided an opportunity to determine if E93 binds to the salivary gland polytene chromosomes and, if so, to identify the sites bound by the protein. Salivary glands were dissected 12-14 hr after puparium formation, fixed, squashed, and photographed to acquire accurate cytology of the banding and puffing patterns for mapping. The chromosomes were then stained with affinity-purified E93 antibodies, and these patterns were compared with the original set of photographs to allow accurate mapping of the bound sites. E93 clearly binds to the polytene chromosomes in a reproducible and site-specific manner and is consistently detected at 65 chromosome sites, many of which contain ecdysone-regulated genes or programmed cell death genes. Among these sites are the 74EF and 75B early puffs, which contain the E74 and E75 ecdysone-inducible genes, as well as the 93F puff, which contains E93. In addition, 1B, 21C, 59F, and 99B are bound by E93 and contain the programmed cell death genes dredd, crq, dcp-1, and drICE, respectively. The 2B5 early puff, containing the BR-C ecdysone-inducible gene, and 75CD, containing βFTZ-F1 and the programmed cell death genes rpr, hid, and grim, were not bound by E93. These data indicate that E93 may directly regulate the genes in bound chromosome loci and may either encode a site-specific DNA binding protein or a chromatin-associated protein that functions as a transcriptional regulator (Lee, 2000).

The observations that E93 is essential for salivary gland cell death and that E93 protein binds to specific sites in the salivary gland polytene chromosomes suggest that E93 may regulate the transcription of target genes that function in steroid-triggered programmed cell death. If this hypothesis is true, then E93 mutations should impact the transcription of genes that reside in salivary gland chromosome loci bound by E93. Salivary glands were dissected from staged late third instar larvae, prepupae, and pupae of control and mutant animals. Total RNA extracted from these tissues was analyzed by Northern blot hybridization. E93 mutations have little or no effect on the timing and levels of BR-C, E74, and E75A transcription in the salivary glands of late third instar larvae and early prepupae. However, the level of expression of each of these regulatory genes is significantly reduced or absent in salivary glands 10-24 hr following puparium formation. Although the smaller E74B transcript is induced, the larger E74A RNA is not detected following the prepupal pulse of ecdysone. The levels of EcR expression in late third instar larval and prepupal salivary glands are not altered by E93 mutations, although its timing is delayed by 4-6 hr at the prepupal to pupal transition. Like EcR, βFTZ-F1 transcription is delayed but the level of this mRNA is not altered in E93 mutant salivary glands. A similar delay is observed in the parental flies that were used for mutagenesis, indicating that this effect is due to the genetic background. The induction of EcR and E74B in E93 mutant prepupae, as well as the successful completion of adult head eversion, indicates that the prepupal pulse of ecdysone occurs in these mutant animals, signaling the prepupal-pupal transition (Lee, 2000).

E93 mutant salivary glands also exhibited little or no transcription of genes that play a key role in programmed cell death. rpr and hid are induced in control animals in a stage-specific manner, immediately preceding the onset of salivary gland cell death. Interestingly, the relative of the vertebrate CD36 gene named croquemort (crq), ark, and the caspase dronc are also induced at this time, indicating that other components of the apoptotic signaling pathway are utilized during programmed cell death in salivary glands. Transcription of the caspases dredd, dcp-1, and drICE is not detected in salivary glands at these developmental stages. The cell death genes rpr, hid, crq, ark, and dronc are transcribed at reduced levels in E93 mutant salivary glands 12-24 hr following puparium formation. These observations indicate that E93 functions as a key regulator by specifying the steroid activation of cell death genes (Lee, 2000).

Programmed cell death (PCD), important in normal animal physiology and disease, can be divided into at least two morphological subtypes, including type I, or apoptosis, and type II, or autophagic cell death. While many molecules involved in apoptosis have been discovered and studied intensively during the past decade, autophagic cell death is not well characterized molecularly. This study reports the first comprehensive identification of molecules associated with autophagic cell death during normal metazoan development in vivo. During Drosophila metamorphosis, the larval salivary glands undergo autophagic cell death regulated by a hormonally induced transcriptional cascade. To identify and analyze the genes expressed, wild-type patterns of gene expression were examined in three predeath stages of Drosophila salivary glands using serial analysis of gene expression (SAGE). 1244 transcripts, including genes involved in autophagy, defense response, cytoskeleton remodeling, noncaspase proteolysis, and apoptosis, were seen to be expressed differentially prior to salivary gland death. Mutant expression analysis indicated that several of these genes were regulated by E93, a gene required for salivary gland cell death. These analyses strongly support both the emerging notion that there is overlap with respect to the molecules involved in autophagic cell death and apoptosis, and that there are important differences (Gorski, 2003).

Distinct promoter regions regulate spatial and temporal expression of the Drosophila caspase dronc: E93 targets dronc

Dronc is an apical Drosophila caspase essential for programmed cell death during fly development. During metamorphosis, dronc gene expression is regulated by the steroid hormone ecdysone, which also regulates the levels of a number of other critical cell death proteins. As dronc protein levels are important in determining caspase activation and initiation of cell death, the regulation of the dronc promoter was analyzed using transgenic flies expressing a LacZ reporter gene under the control of the dronc promoter. These results indicate that dronc expression is highly dynamic during Drosophila development, and is controlled both spatially and temporally. While a 2.3 kb dronc promoter region contains most of the information required for correct gene expression, a 1.1 kb promoter region is expressed in some tissues and not others. During larval-pupal metamorphosis, two ecdysone-induced transcription factors, Broad-Complex and E93, are required for correct dronc expression. These data suggest that the dronc promoter is regulated in a highly complex manner, and provides an ideal system to explore the temporal and spatial regulation of gene expression driven by nuclear hormone receptors (Daish, 2003).

Experiments outlined in this paper demonstrate that 2.3 kb of the dronc promoter is largely sufficient for temporal expression (compared to endogenous dronc) throughout development. Previous experiments have shown that dronc is predominantly expressed in the larval and prepupal salivary glands and midgut, and larval brain lobes. 2.3 kb of the dronc promoter contains all necessary elements for correct spatial regulation of dronc expression in these tissues (Daish, 2003).

In order to identify transcription factors responsible for both temporal and spatial regulation of dronc and ecdysone-mediated PCD, it is of vital importance to elucidate the regions of the promoter essential for dronc expression in different tissues. In addition, it would be of interest to determine if there is a single promoter region controlling the spatial expression profile of dronc, or if different promoter regions are required in different tissues. LacZ transgenic reporter experiments reveal that the 2.3 kb promoter is the minimal requirement for correct expression in brain lobes and salivary glands. Furthermore, the region between 1.1 and 2.3 kb contains transcription factor-binding sites essential for expression in these tissues. This region also seems to harbor a repressor element important to keep dronc levels low during periods when ecdysone titers are low. Surprisingly, regulation of dronc transcription is markedly different in the midgut. The region between 1.1 and 2.3 kb is not important for transcription in this tissue, because 1.1 kb of the promoter is sufficient for expression. These results clearly demonstrate that distinct regions of the promoter are required for expression in different tissues, and implies that different transcription factors regulate dronc expression in a tissue-dependent manner (Daish, 2003).

The two ecdysone-induced transcription factors BR-C and E93 are essential for dronc expression in salivary glands. In the midgut, however, only E93 seems to be important. The results of dronc promoter-LacZ transgenic expression in flies deficient in BR-C and E93 are consistent with recent findings. LacZ expression driven by the 2.8 kb promoter is severely impaired in salivary glands of BR-C (rbp5 and npr) or E93 mutants, whereas expression is impaired only in the midgut of E93 mutant background animals. This further supports the idea that the mechanisms governing dronc regulation are tissue specific. The key questions arising from these experiments are: why does the BR-C Z1 isoform (rbp5 mutant) regulate dronc in the salivary glands and not in the midgut? What factors are binding to the 1.1-2.3 kb region of the promoter in salivary glands, and why are they not as important in the midgut? Previous results show that either BR-C Z1- or BR-C Z1-regulated proteins bind to the dronc proximal promoter and control its expression. Transactivation of the 2.8 kb promoter by BR-C Z1, however, was only seen in specific cell types. Given that BR-C Z1 is also expressed in the midgut, this implies that it may be acting through cofactors which are not expressed in the midgut, yet are specifically recruited to the dronc promoter. Alternatively, BR-C Z1 induces the expression of another factor which binds to the promoter, and this factor is absent in the midgut (Daish, 2003).

Since the proximal promoter alone (0.54 kb) is not sufficient for expression in the salivary gland, it is believed that BR-C Z1 (or a Z1-regulated protein) is cooperating with other transcription factors binding upstream (1.1-2.3 kb), that are essential for salivary gland expression. It has been shown that E93 acts through the first 600 bp of the dronc promoter by transactivation studies; however, no direct binding of E93 to the dronc (or any other) promoter has been shown so far. Additionally, a preliminary analysis indicates the presence of an EcR/Usp-binding site between 1.1 and 2.3 kb of the dronc promoter, and in vitro experiments show that this element may be important in regulating dronc expression. Since the proximal promoter (0.54 kb) alone is not sufficient for expression, cooperation of BR-C and E93 with EcR/Usp and other unknown factors may be important for temporal and spatial regulation of dronc expression during development. Identification of these factors will be important for fully understanding dronc transcription during development (Daish, 2003).

Overall, this study has established the minimal dronc promoter requirement for spatial and temporal expression to be within the 2.3 kb region upstream of the dronc gene. This region is important for both BR-C- and E93-mediated transcription in salivary glands and E93 transcription in the midgut. Importantly, the 1.1-2.3 kb promoter region harbors elements important for salivary gland expression and a putative repressor element. The 0.54-1.1 kb promoter region is important for expression in the midgut. These regions will form the basis of future experiments designed to identify factors necessary for the regulation of dronc expression during PCD (Daish, 2003).

DEVELOPMENTAL BIOLOGY

Larval and Pupal Phases

Pulses of ecdysteroids induce dramatic changes in gene expression that direct the early stages of Drosophila metamorphosis. This gene activity is reflected by the appearance of early and late puffs in the salivary gland polytene chromosomes. Curiously, the early puff genes that have been studied to date are induced by both the late larval and prepupal pulses of ecdysteroids and are expressed in many ecdysteroid target tissues, raising the question of how the hormone directs the complex stage- and tissue-specific responses associated with metamorphosis. In an effort to address this question, the E93 gene responsible for the stage-specific 93F early puff has been isolated and characterized. The E93 mRNA displays no response to ecdysteroids in late larval salivary glands but is directly induced 12 hr later by the prepupal ecdysteroid pulse, identical to the response of the 93F puff. E93 transcripts are first detected during metamorphosis on Northern blots. Transcript levels increase following the late larval and prepupal ecdysteroid pulses, as well as immediately before adult eclosion. In tissues other than the salivary gland, however, E93 displays complex spatial and temporal regulation. In situ hybridization and Northern blot analysis of RNA isolated from dissected tissues shows that E93 transcripts are present in gut and at a lower level in fat body of early prepupae and in the CNS, gut , fat body, salivary glands and imaginal discs in late prepupae (Baehrencke, 1995).

To gain further insight into the cellular distribution and potential biochemical function of this protein, antibodies were made against E93. Staged midguts and salivary glands were stained with affinity-purified E93 antibodies to determine the spatial and temporal patterns of E93 expression. E93 is not detected in the midguts of late third instar larvae but is expressed in the midguts of newly formed prepupae, paralleling the induction of E93 mRNA at puparium formation. E93 is detected immediately prior to the destruction of midgut gastric caeca and midgut shortening, both of which coincide with the onset of programmed cell death. Interestingly, E93 is not expressed in the diploid cells that form the adult midgut epithelium. Larval salivary glands do not express E93 in early and mid prepupae. Following the pulse of ecdysone in 10-12 hr prepupae, however, E93 expression is induced in salivary glands, reflecting the induction of E93 mRNA at this time and foreshadowing programmed cell death (Jiang, 1997). E93 is not expressed in leg and wing imaginal discs during prepupal development but is detected in a subset of cells in the developing eye and central nervous system. These results are consistent with the hypothesis that E93 is expressed in dying cells, since the eye and central nervous system undergo programmed cell death at this developmental stage. In addition, E93 is restricted to the nucleus, suggesting that E93 might regulate gene expression (Lee, 2000).

EFFECTS OF MUTATION

An F2 lethal screen was performed to isolate ethane methyl sulfonate (EMS)-induced mutations in E93. From a total of 11,134 F2 EMS-mutagenized lines, 29 lines were isolated that possess lethal mutations within the region defined by Df(3R)93F^X2, which removes E93. These mutations define 11 lethal complementation groups. Two of these complementation groups display pupal lethal phenotypes, while the other nine result in lethality at earlier stages in development. One of the pupal lethal complementation groups, represented by three alleles, dies late during metamorphosis with defects that are restricted to developing adult structures. The second pupal lethal complementation group, also represented by three alleles, dies earlier during pupal development. Because this lethal phase corresponds to the earliest expression of E93, these mutants were subjected to more detailed phenotypic and molecular characterization. The 3.6 kb E93 open reading frame, as well as intron/exon boundaries, were sequenced from genomic DNA isolated from each of the three mutant alleles as well as the parental strain used for mutagenesis. The E93¹ allele has a T-to-A transition at nucleotide 3374 that changes a leucine at position 994 to a stop codon. While no mutations were detected in either E93² or E93³, a significantly reduced amount of E93 mRNA was detected in homozygous E93² mutants, and no E93 mRNA was detected in homozygous E93³ mutants. These results indicate that the E93² and E93³ mutations affect either transcriptional regulatory elements or E93 mRNA stability. Taken together, these results suggest that E93¹, E93², and E93³ represent either strong hypomorphic or null E93 mutations (Lee, 2000).

E93 mutants display little lethality during embryonic and larval development and die during the early stages of pupal development. These mutants fail to shorten their body properly at puparium formation, often exhibit a defect in anterior spiracle eversion, and die following head eversion. Although E93 mutants possess a well-developed head, thorax, and abdomen, no pigmentation of adult structures occurs, even following prolonged aging. E93¹ mutants exhibit identical phenotypes when combined with a deletion of E93 or as homozygotes and, therefore, fulfill the genetic definition of a null allele. Similarly, E93² and E93³ were shown to behave as strong loss-of-function or null alleles and exhibit identical phenotypes when transheterozygous with the E93¹ allele (Lee, 2000).

In order to gain a better understanding of the developmental defects associated with E93 mutations, animals were staged 24 hr following puparium formation, embedded in paraffin, and sectioned. At this developmental stage, control animals have formed adult structures, including eyes and wings, and the larval salivary glands have been completely destroyed. In contrast, E93 mutants possess persistent larval salivary glands, even though adult structures have formed, including eyes and wings. This defect in salivary gland death is completely penetrant in all three E93 mutant alleles. Furthermore, mutant salivary glands can be detected for days after they would normally be destroyed. In addition to the salivary gland defect, E93 mutants display defects in larval midgut destruction. The observation that salivary gland cell death is blocked in E93 mutants while adult head eversion occurs normally indicates that these animals have progressed through the ecdysone-regulated prepupal-pupal transition with specific defects in the destruction of larval cells (Lee, 2000).

If the defects in salivary gland cell death are caused by mutations in E93, then this phenotype should be rescueable by ectopic expression of E93. For this purpose, a transgenic fly stock was established in which the E93 gene is under control of the yeast GAL4 upstream activation sequence. This UAS-E93 construct was combined with a GAL4 transgene expressed in salivary glands in an E93¹/Df(3R)93F^X2 genetic background. In all cases examined, E93 mutants that carry the UAS-E93 transgene lacked salivary glands, while sibling controls that lack the transgene possess salivary glands. This observation indicates that E93 is required in the salivary gland for its appropriate programmed cell death response (Lee, 2000)

Apoptosis and autophagy are morphologically distinct forms of programmed cell death. While autophagy occurs during the development of diverse organisms and has been implicated in tumorigenesis, little is known about the molecular mechanisms that regulate this type of cell death. Steroid-activated programmed cell death of Drosophila salivary glands occurs by autophagy. Expression of p35 prevents DNA fragmentation and partially inhibits changes in the cytosol and plasma membranes of dying salivary glands, suggesting that caspases are involved in autophagy. The steroid-regulated BR-C, E74A and E93 genes are required for salivary gland cell death. BR-C and E74A mutant salivary glands exhibit vacuole and plasma membrane breakdown, but E93 mutant salivary glands fail to exhibit these changes, indicating that E93 regulates early autophagic events. Expression of E93 in embryos is sufficient to induce cell death with many characteristics of apoptosis, but requires the H99 genetic interval that contains the rpr, hid and grim proapoptotic genes to induce nuclear changes diagnostic of apoptosis. In contrast, E93 expression is sufficient to induce the removal of cells by phagocytes in the absence of the H99 genes. These studies indicate that apoptosis and autophagy utilize some common regulatory mechanisms (Lee, 2001).

Morphological studies of developing vertebrate embryos have resulted in the definition of three types of physiological cell death. The first type, widely known as apoptosis, is found in isolated dying cells that exhibit condensation of the nucleus and cytoplasm, followed by fragmentation and phagocytosis by cells that degrade their contents. The second type, known as autophagy, is observed when groups of associated cells or entire tissues are destroyed. These dying cells contain autophagic vacuoles in the cytoplasm that function in the degeneration of cell components. Autophagic cells destroy their own contents, while apoptotic cells depend on phagocytes to accomplish terminal degradation. The third type, known as non-lysosomal cell death, is least common, and is characterized by swelling of cavities with membrane borders followed by degeneration without lysosomal activity. While autophagy fulfills the definition of programmed cell death, occurs during development of diverse organisms, and has been implicated in tumorigenesis, little is known about the molecular genetic mechanisms underlying this type of programmed cell death. The morphological characteristics that distinguish apoptosis and autophagy suggest that these cell deaths are regulated by independent mechanisms. Comparison of biochemical changes during lymphocyte apoptosis and insect intersegmental muscle autophagy also indicate that these physiological cell deaths occur by distinct mechanisms. However, recent studies of steroid-triggered cell death of Drosophila larval salivary glands suggest that these cells utilize genes that are part of the conserved apoptosis pathway, even though these cells exhibit characteristics of autophagy. Specifically, the caspase Dronc and the homolog of ced4/Apaf-1 (Ark), two components of the core apoptotic machinery, increase in transcription immediately prior to salivary gland cell death. Thus, characterization of the mechanisms governing the regulation of autophagy will identify how these cell deaths differ from those that occur by apoptosis (Lee, 2001 and references therein).

Larval salivary glands of Drosophila undergo rapid programmed cell death in response to ecdysone. This cell destruction can be detected using markers that are typically associated with apoptosis including nuclear staining by Acridine Orange, TUNEL to detect DNA fragmentation, and exposure of phosphatidylserine on the outer leaflet of the plasma membrane. The changes in vacuolar structure that immediately precede the synchronous destruction of larval salivary gland cells are clearly more similar to autophagy than heterophagy (apoptosis). Large vacuoles increase in number in prepupal salivary glands, and rearrangement of the cytoskeleton and an increase in acid phosphatase activity are associated with these structures. Dynamic changes in salivary gland structure may reflect important biochemical changes during programmed cell death. Large Eosin-positive vacuoles appear to fragment, a distinct class of Eosin-negative vacuoles are formed that are closely associated with the plasma membrane, and vacuoles containing organelles are observed in the cytoplasm immediately preceding destruction of salivary glands. An increase in transcription of the caspase Dronc occurs at this stage, and inhibition of caspase activity blocks DNA fragmentation and partially prevents changes in vacuoles and plasma membranes, suggesting that these morphological changes may be attributed in part to the activity of enzymes typically associated with apoptosis (Lee, 2001 and references therein).

While morphological analyses of apoptosis and autophagy suggest different mechanisms for these forms of cell death, some genes that function in apoptosis also function during autophagy. Steroid-regulated genes impact distinct cellular changes in dying cells. Ecdysone impacts on the transcription of the cell death genes rpr, hid and diap2. This regulation is mediated by the ecdysone receptor, and a group of ecdysone-activated factors that include the BR-C, E74 and E93 genes. The function of the steroid-regulated BR-C, E74 and E93 genes in salivary gland cell death has been examined. E93 mutant salivary glands exhibit persistence of large vacuoles and plasma membranes, while these structures are destroyed in BR-C and E74A mutants. Two possible explanations exist for the differences in BR-C, E74A and E93 mutant salivary gland cell morphology. E93 mutant salivary glands could be arrested at an earlier stage of cell destruction that is similar to that of 12-hour wild-type cells, while BR-C and E74A mutants are arrested at a stage that is similar to 14.5-hour salivary gland cells. This model is supported by previous studies indicating that E93 function is required for proper regulation of BR-C and E74A transcription. Alternatively, E93 could function to regulate autophagy that results in destruction of vacuoles and plasma membranes, while BR-C and E74A do not function in the regulation of these cellular changes even though these genes are required for salivary gland cell death. The latter interpretation is intriguing when one considers that expression of E93 is sufficient to induce characteristics of apoptosis, and can induce the removal of cells even in the absence of the rpr, hid and grim cell death genes and nuclear apoptotic changes (Lee, 2001).

Several factors indicate that salivary gland autophagy is regulated by genes that also function in apoptosis. (1) Caspases function in salivary gland cell death. Expression of the baculovirus inhibitor of caspases, p35, inhibits destruction of this tissue. Furthermore, p35 expression prevents DNA fragmentation and partially inhibits morphological changes in vacuoles that are associated with autophagy, indicating that caspases are utilized during autophagy. Transcription of the Apaf1 homolog Ark and the caspase, dronc increases immediately preceding salivary gland cell death, and this transcription is blocked in E93 mutants, further supporting that caspases function in salivary gland autophagy. (2) Transcription of the proapoptotic genes, rpr and hid increases immediately prior to salivary gland autophagy, and the transcription of these genes is blocked by mutations in steroid-regulated genes that are involved in this process. Ectopic expression of E93, a critical determinant of salivary gland autophagy, is sufficient to induce cell death with numerous characteristics of apoptosis. In addition, the association of Croquemort (Crq) expression with E93-induced removal of apoptotic cells and autophagy of salivary glands provides yet another link between these morphologically distinct forms of programmed cell death. Combined, these factors indicate that autophagy and apoptosis utilize at least some similar mechanisms (Lee, 2001).

The location and type of cell appears to be an important determinant for the type of programmed cell death that occurs in the context of animal development. Autophagy occurs when groups of cells or entire tissues die, while apoptosis occurs in isolated dying cells. These studies are consistent with these criteria; salivary gland destruction occurs by autophagy and requires E93 function, while ectopic induction of cell death by expression of E93 during embryogenesis has the characteristics of apoptosis. It is hypothesized that this is due to similarities between autophagy and apoptosis. Alternatively, autophagy and apoptosis may be mechanistically distinct, and the ability to induce ectopic cell death by expression of E93 is simply due to activating a death program in different cell types. This explanation is supported by data demonstrating that p35 inhibits salivary gland cell death, but that p35 is not capable of inhibiting E93-induced cell death in embryos. However, several possibilities exist to explain the disparity of these data. (1) Ectopic expression of E93 during embryogenesis may lead to higher than normal levels of this protein. In side-by-side comparisons with the proapoptotic genes rpr and hid, expression of E93 results in greater cell death and lethality. Thus, the strong killing potential of E93 may be sufficient to overcome inhibition of cell death by p35. (2) Other cell death genes are not inhibited by expression of p35, including cell death that is induced by ectopic expression of the caspase Dronc. (3) Inhibition of vacuolar changes by expression of p35 during salivary gland cell death is incomplete, even though DNA fragmentation is inhibited in this tissue. Thus, caspases may play a role in salivary gland cell death, and both p35 experiments and the transcription of dronc during salivary gland autophagy support this conclusion. However, it is possible that other proteolytic mechanisms act in concert with caspases in the bulk degradation of salivary gland cells (Lee, 2001).

It is concluded that autophagy and apoptosis are morphologically distinct, suggesting that the mechanisms underlying the regulation of these forms of programmed cell death are different. Nearly all of the large polytenized larval cells die during Drosophila metamorphosis. The synchrony and volume of these cell deaths suggests that engulfment of each dying cell may be limited by the number of available phagocytes. One obvious distinction between autophagy and apoptosis is the location of the lysosomal machinery that degrades the dying cell. Autophagic cells destroy their own contents, while apoptotic cells depend on phagocytes to accomplish terminal degradation. This distinction may account for much of the differences in the morphological appearance of these two forms of dying cells, but does not exclude the possibility that a single autophagic cell utilizes the mechanisms that exist in distinct apoptotic and phagocytic cells. The specific expression of Crq during autophagy supports this possibility, but genetic studies of crq function are needed to test this hypothesis. Future studies of autophagy, and its relationship to apoptosis, will illustrate the similarities and differences between these forms of programmed cell death (Lee, 2001).

Apoptosis and autophagy are two forms of programmed cell death that play important roles in the removal of unneeded and abnormal cells during animal development. While these two forms of programmed cell death are morphologically distinct, recent studies indicate that apoptotic and autophagic cell death utilize some common regulatory mechanisms. To identify genes that are associated with apoptotic and autophagic cell death, changes in gene transcription were monitored by using microarrays representing nearly the entire Drosophila genome. Analyses of steroid-triggered autophagic cell death identified 932 gene transcripts that changed 5-fold or greater in RNA level. In contrast, radiation-activated apoptosis resulted in 34 gene transcripts that exhibited a similar magnitude of change. Analyses of these data enabled identification of genes that are common and unique to steroid- and radiation-induced cell death. Mutants that prevent autophagic cell death exhibit altered levels of gene transcription, including genes encoding caspases, non-caspase proteases, and proteins that are similar to yeast autophagy proteins. This study also identifies numerous novel genes as candidate cell death regulators and suggests new links between apoptosis and autophagic cell death (Lee, 2003).

The identification of genes that exhibit significant changes in RNA levels during steroid-triggered autophagic cell death and radiation-induced apoptosis prompted empirical analyses of transcription in mutants that block salivary gland cell death. Mutations in the ecdysone-regulated genes BR-C, E74A, and E93 prevent salivary gland programmed cell death and prevent proper transcription of the apoptosis genes rpr, W (hid), ark, Nc (dronc), and crq. The transcription of a subset of the newly identified genes was examined in BR-C, E74A, and E93 mutants by Northern blot hybridization because of their possible association with apoptosis and autophagy in dying salivary glands. Cohybridization of these Northern blots allows systematic investigation of how BR-C, E74A, and E93 might regulate transcription of genes that were identified with Genechips and provides a possible mechanism to explain steroid regulation of cell death (Lee, 2003).

The radiation-inducible genes CG10965, CG17323, CG7144, EG25E8.4, and CG5254 are induced in control dying salivary glands at head eversion, and this transcription is altered in mutants that prevent salivary gland cell death. CG10965 and CG17323 are not transcribed in salivary glands of BR-C mutants; they exhibit elevated levels of transcription in E74A mutants, and have reduced RNA levels in E93 mutants. CG7144 is transcribed at significantly reduced levels in BR-C mutants, is ectopically transcribed before the rise in ecdysone in salivary glands of E74A mutants, and may also be ectopically transcribed in E93 mutants. EG25E8.4 is not altered in BR-C and E74A mutants, but this RNA is significantly reduced in salivary glands of E93 mutants. CG5254 is not transcribed in BR-C mutants, had normal RNA levels in E74A mutants, and had reduced RNA levels in E93 mutants (Lee, 2003).

Several other categories of genes exhibit interesting patterns of regulation in BR-C, E74A, and E93 mutant salivary glands. The Bcl-2 family member buffy and the caspases Ice (drice) and dream (strica) are induced at head eversion in salivary glands of control animals, and they are altered to different extents in mutants. Similarly, the Drosophila genes that are most similar to the yeast autophagy genes apg2 (CG1241), apg4 (CG6194), apg5 (CG1643), apg7 (CG5489), and apg9 (CG3615) are induced just prior to cell death of wild-type salivary glands, and they are altered to varying extents in BR-C, E74A, and E93 mutants. It is particularly intriguing that E93 mutants have significantly decreased levels of CG6194, CG1643, and CG5489, since yeast with mutations in apg4, apg5, and apg7 are defective in autophagosome formation and size, and E93 mutants exhibit defects in vacuolar changes in dying salivary gland and midgut cells. In addition, the cysteine protease (CG5505), serine protease (CG3650), and metalloprotease (mmp1) all exhibit increases in RNA level immediately following the rise in ecdysone in dying wild-type salivary glands, and this change is accompanied by a decrease in the inhibitor of metalloproteases, timp. It is interesting that BR-C, E74A, and E93 mutations affect transcription of the non-caspase protease genes CG5505, CG3650, and mmp1, since caspase inhibitors do not completely block changes in dying salivary glands, and mutations in these ecdysone-regulated genes prevent degradation of salivary gland cells (Lee, 2003).

Drosophila salivary gland chromosomes were used to predict the first steroid-triggered transcription hierarchy based on chromosome puffing (chromatin decondensation). This study has identified several candidate genes in this signaling pathway based on correlative increases in transcription that are associated with chromosome puffs and with the proximity of binding sites of transcription factors in this pathway. Two putative puff genes, CG17309 (86E puff) and CG3132 (87A puff), increase following the rise in ecdysone titer and match the puffing patterns of these chromosome loci. CG17309 RNA is present before the rise in ecdysone in BR-C mutants, while it is reduced in salivary glands of E74A and E93 mutants. CG3132 appears to encode two transcription units that were either not detected or decreased in salivary glands of BR-C, E74A, and E93 mutants. The Smad anchor for receptor activation sara and the transcription regulator bun have increased RNA levels in dying salivary glands and have BR-C Z1 and E74A binding sites in the same region of the genome. sara is not induced in BR-C, E74A, and E93 mutant salivary glands. bun RNA was also not detected in BR-C and E93 mutant salivary glands, but it is expressed normally in E74A mutant salivary glands. These data provide a direct link between the ecdysone-regulated early genes and target genes (Lee, 2003).

It is concluded that developmental cues and genotoxic stress can both trigger programmed cell death. During steroid-triggered autophagic cell death in developing salivary glands, 932 gene transcripts were identified that either decreased or increased 5-fold or greater in RNA level. In contrast, radiation-activated apoptosis in embryos only identified 34 gene transcripts that exhibited a similar magnitude of change. The difference in the number of genes that were induced by these stimuli most likely reflects the presence of maternal RNAs for cell death genes that are deposited in embryos. Alternatively, the apoptotic machinery may exist in cells as proteins waiting to be posttranslationally activated following a death-inducing stimulus. Radiation-induced apoptosis in Drosophila embryos can be suppressed by treatment with cyclohexamide, suggesting that protein synthesis is necessary for activation of this cell death. In addition, studies of radiation-induced apoptosis have implicated p53, which is known to function as a regulator of transcription in this process. It is also possible that radiation-induced apoptosis is sufficiently asynchronous that it is difficult to detect changes in RNA levels in a very complex cell population. Comparative analyses of cell death microarray data has enabled the identification of a small group of genes that are induced by both ecdysone and radiation. While salivary gland autophagic cell death and radiation-induced apoptosis appear to be quite different, transcription of the common genes rpr, CG10965, CG17323, CG7144, EG25E8.4, and CG5254 is altered in mutants that prevent salivary gland cell death, further suggesting that these genes are important for this cell death. In addition, BR-C, E74A, and E93 mutants also impact transcription of numerous genes in salivary glands, including apoptosis regulators, non-caspase proteases and protease inhibitors, cell remodeling factors, and the genes that are similar to the yeast genes that function in protein degradation by autophagy. This study has identified numerous genes that exhibit interesting patterns of transcription during steroid- and radiation-induced programmed cell death, and future genetic studies will determine the importance of these genes in autophagy and apoptosis (Lee, 2003).

Self-digestion of cytoplasmic components is the hallmark of autophagic programmed cell death. This auto-degradation appears to be distinct from what occurs in apoptotic cells that are engulfed and digested by phagocytes. Although much is known about apoptosis, far less is known about the mechanisms that regulate autophagic cell death. Autophagic cell death is regulated by steroid activation of caspases in Drosophila salivary glands. Salivary glands exhibit some morphological changes that are similar to apoptotic cells, including fragmentation of the cytoplasm, but do not appear to use phagocytes in their degradation. Changes in the levels and localization of filamentous Actin, alpha-Tubulin, alpha-Spectrin and nuclear Lamins precede salivary gland destruction, and coincide with increased levels of active Caspase 3 and a cleaved form of nuclear Lamin. Mutations in the steroid-regulated genes ßFTZ-F1, E93, BR-C and E74A that prevent salivary gland cell death possess altered levels and localization of filamentous Actin, alpha-Tubulin, alpha-Spectrin, nuclear Lamins and active Caspase 3. Inhibition of caspases, by expression of either the caspase inhibitor p35 or a dominant-negative form of the initiator caspase Dronc, is sufficient to inhibit salivary gland cell death, and prevent changes in nuclear Lamins and alpha-Tubulin, but not to prevent the reorganization of filamentous Actin. These studies suggest that aspects of the cytoskeleton may be required for changes in dying salivary glands. Furthermore, caspases are not only used during apoptosis, but also function in the regulation of autophagic cell death (Martin, 2004).

Studies of salivary glands indicate that caspases play an important role in their autophagic cell death. The caspase-encoding genes dronc and drice show an increase in their transcription following the rise in steroid that triggers salivary gland autophagic cell death. This increase in caspase transcription corresponds to the increase in active caspase protein levels and in the cleavage of substrates such as nuclear Lamins in dying salivary glands. Mutations in the steroid-regulated ßFTZ-F1, E93 and BR-C genes, which prevent salivary gland cell death, exhibit little or no active Caspase-3/Drice expression, and have altered alpha-Tubulin, alpha-Spectrin and nuclear Lamin expression in salivary glands. Although E74A mutants prevent salivary gland cell death, they have elevated Caspase-3/Drice levels and degraded nuclear Lamins. Although these data are consistent with the partially degraded morphology of E74A mutant salivary glands, it remains unclear what factor(s) E74A may regulate that are required for normal cell death. However, the data indicate that ßFTZ-F1, E93 and BR-C play a crucial role in determining caspase levels in dying salivary gland cells, and this is supported by the impact of these genes on the transcription of dronc. Significantly, inhibition of caspases by expression of either p35 or dominant-negative Dronc is sufficient to prevent DNA fragmentation, changes in nuclear Lamins and alpha-Tubulin, and death of salivary glands (Martin, 2004).

E93 regulates the autophagic death of midgut cells during 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).

The morphology of midguts was examined at the onset of metamorphosis to provide a framework for studies of genetic regulation of larval midgut cell death. Wild-type Canton S were staged in hours following puparium formation, fixed, embedded in paraffin, sectioned, and stained. New prepupae possess a larval esophagus, proventriculus, gastric caeca, and midgut structures, and exhibit no signs of larval cell death or adult midgut formation at this resolution. Two hours after puparium formation, the proventriculus, gastric caeca, and larval midgut are surrounded by an adult epithelium. In 4-h prepupae, the proventriculus and gastric caeca appear to compress toward the larval midgut, and the larval epithelium becomes convoluted, causing a large space in the larval lumen. Six hours after puparium formation, the proventriculus and gastric caeca can no longer be distinguished, and the larval midgut becomes further condensed. The larval midgut is extremely condensed 12 h after puparium formation, and the adult and larval epithelia have separated such that a defined adult lumen exists (Lee, 2002).

The death of larval midgut cells coincides with the increase in ecdysone that triggers puparium formation, and premature elevation of the ecdysone titer in third instar larvae is sufficient to ectopically induce cell death in larval midguts. In addition, mutations in the Ecdysone receptor and the ecdysone-regulated primary response gene BR-C prevent proper destruction of larval midguts. The role of the ecdysone-regulated primary response genes BR-C, E93, and E74 in larval midgut destruction was examined, since these genes regulate steroid-activated destruction of larval salivary glands (Lee, 2002).

To analyze the destruction of mutant larval midguts, animals were staged at pupal head eversion, fixed, embedded in paraffin, sectioned, and analyzed by light microscopy for defects in midgut structure. Head eversion was selected as the stage for analyses since this is 12 h after midgut destruction is initiated and the larval midguts of control animals are fully compressed at this time. BR-C (2Bc²) mutants have the strongest phenotype and always possess some remnants of the larval proventriculus and gastric caeca. While larval midgut destruction does not occur properly in BR-C mutants, the adult epithelium is formed and the midgut appears to be arrested at a stage that is similar to the midgut of wild-type animals 2-4 h following puparium formation (Lee, 2002).

E93 mutants always form an adult epithelium, and the larval proventriculus and gastric caeca are destroyed. However, larval midgut compaction is never observed in E93 mutants as indicated by the large space in the larval lumen. E93 mutant larval midguts appear to be arrested at a stage of destruction that is similar to the midgut of wild-type animals 4-6 h following puparium formation. E74A and E74B mutants also form an adult midgut epithelium, and the larval proventriculus and gastric caeca are destroyed. Larval midgut compaction seems to occur to a greater extent in E74A and E74B mutants than in E93 mutants, since E74A and E74B mutants develop a large space between the larval and adult midgut epithelia (Lee, 2002).

Thus, E74A and E74B mutant larval midguts appear to be arrested at a stage of destruction that is similar to the midgut of wild-type animals 6-12 h following puparium formation and do not appear to impact larval midgut destruction (Lee, 2002).

The larval midgut shrinks dramatically during the first 6 h of pupariation when these cells are dying, suggesting that midgut shortening may be related to larval cell death. In order to quantify the relationship between the change in midgut length, cell death, and mutants that impact larval midgut destruction, the length of midguts was measured at puparium formation and head eversion. Wild-type Canton S midguts decrease from 7.80 to 1.60 mm in length, or 85%, during this interval. While BR-C, E93, E74A, and E74B mutant larval midguts all decrease in size between puparium formation and head eversion, this shrinking varies. BR-C mutant midguts decrease from 7.57 to 2.50 mm (65%) in length. E93 midguts decrease from 6.38 to 1.64 mm (74%) between puparium formation and head eversion. Midguts of E74A mutants decrease from 6.55 to 1.21 mm (81%), while E74B mutants change from 5.97 to 1.62 mm (73%) in length. Therefore, midgut shortening likely relies on the newly formed adult midgut epithelium, since midguts shorten in BR-C and E93 mutants that prevent proper destruction of larval cells (Lee, 2002).

DNA fragmentation accompanies the destruction of larval midguts. The TUNEL procedure was used to determine whether mutations in ecdysone-regulated genes prevent DNA fragmentation in larval midgut cells. Wild-type Canton S and mutant animals were staged at head eversion, fixed, embedded in paraffin, sectioned, and analyzed for the presence or absence of DNA fragmentation. Canton S possess compacted midguts and fragmented DNA at head eversion. While BR-C mutants have persistent larval structures, including gastric caeca, every larval midgut cell nucleus of these mutants appeared to be labeled, indicating that they possess fragmented DNA (Lee, 2002).

Similarly, the nuclei of E93, E74A, and E74B mutant midguts were all labeled following the TUNEL procedure. These data suggest that larval midgut cells do not die by apoptosis, since mutations in the BR-C and E93 genes prevent destruction of midgut cells, and the midgut cells of these mutants possess fragmented DNA (Lee, 2002).

Drosophila larval midguts exhibit markers of apoptosis immediately prior to destruction, including DNA fragmentation and nuclear staining by acridine orange, as well as increased transcription of the proapoptotic genes rpr and hid. While mutations in the BR-C and E93 genes prevent destruction of midgut cells, the midgut cells of these mutants possess fragmented DNA, suggesting that they do not die by apoptosis. Thus, transmission electron microscopy was used to analyze cell structure during cell death of midguts. Late third instar larval midguts possess microvilli facing the lumen, large nuclei with banded polytene chromosomes, and intact mitochondria in the cytoplasm. At this stage, very few indications of cell death exist, although small numbers of early stage autophagic vacuoles and swirls of rough endoplasmic reticulum are observed; this is one of the mechanisms by which autophagic vacuoles are formed. Larval midguts of new prepupae have microvilli facing the lumen, intact nuclei, and the cytoplasm has increased numbers of autophagic vacuoles and appears to possess more spaces than in late third instar larvae. Vacuoles that contain structures, including organelles such as mitochondria and crystalline inclusions, are abundant in the larval midguts of new prepupae and indicate that these cells die by autophagy. Two hours following puparium formation, the forming adult midgut is apparent, and the larval midgut cytoplasm possesses an increased number of vacuoles containing organelles, indicating that autophagy has progressed. Larval midguts of 4-h prepupae appear to exhibit an increase in the number of nuclei per area examined, which is likely due to the compression of this structure. Large numbers of crystalline inclusions were observed in the cytoplasm of larval midguts in 4-h prepupae. The proximity of nuclei increases and autophagic structures are abundant in larval midguts 6 h after puparium formation. Twelve hours after puparium formation, the cytoplasm of larval midguts appears more condensed since fewer spaces are observed, and numerous autophagic structures, including myelin-like membrane swirls, are detected. These data indicate that larval midguts die by autophagy and do not exhibit morphological characteristics of apoptosis (Lee, 2002).

Larval midgut cells possess vacuoles that contain cytosolic structures, such as mitochondria, indicating that these cells die by autophagy. Thus, whether mutations in the BR-C, E93, E74A, and E74B genes prevent the destruction of the cytoplasm was tested. The midgut cells of BR-C mutants exhibit variable cytoplasmic staining: some cells are extremely osmophylic, while others are not stained as dark. BR-C mutant midgut cells contain intact mitochondria and do not exhibit obvious alterations in cytosolic structures from midguts of third instar larvae other than containing large spaces. In contrast, E93 mutant midguts possess numerous cells that contain swollen mitochondria, and many of these organelles rupture. Not all E93 mutant midgut cells completely lack autophagic structures, however, since some mitochondria are enclosed by membranes. The midguts of E74A and E74B mutants contain intact mitochondria that are observed in autophagic vacuoles. While BR-C mutants exhibit defects in the destruction of gross larval structures and E93 mutants exhibit defects in the destruction of cytosolic midgut structures (such as mitochondria), no similar defects were observed in either E74A or E74B mutant midguts, which possess numerous normal autophagic structures (Lee, 2002).

Expression of the caspase inhibitor p35 prevents midgut cell death). Since caspases are generally considered proteases that regulate apoptosis, it was necessary to determine whether caspases and other cell death regulators are transcribed in midguts that die by autophagy. While it is known that rpr and hid are induced in dying midguts, it is not known whether other candidate cell death regulators are induced in these cells. Therefore, developmental Northern blots were prepared from wildtype midguts at stages preceding and during cell death (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).

Studies of ecdysone-triggered destruction of Drosophila larval midguts and salivary glands illustrate many similarities in these dying cells. However, several important differences exist between ecdysone-regulated midgut and salivary gland programmed cell death. Consider that these two tissues are triggered to die by independent pulses of ecdysone. While the nuclear receptor ßFTZ-F1 is responsible for specifying ecdysone induction of BR-C, E74A, and E93 immediately prior to larval salivary gland programmed cell death, the factor(s) that specify the timing of the cell death response in larval midguts 12 h earlier remain unclear. BR-C and E93 appear to be critical regulators of midgut cell death, but it is unclear how the ecdysone receptor complex activates these genes in midguts. ßFTZ-F1 is not expressed in midguts prior to ecdysone-induced cell death of this tissue, so other factors must be responsible for induction of BR-C and E93 in midguts. One possibility is that the hormone receptor complex activates BR-C and E93 independently of a factor such as ßFTZ-F1. Alternatively, another nuclear receptor, or possibly an unrelated transcription regulator, may regulate BR-C and E93. Future genetic studies and analyses of the BR-C and E93 promoters will define the mechanism for the stage-specific induction of cell death by ecdysone in larval midguts (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).

Control of target gene specificity during metamorphosis by the steroid response gene E93

Hormonal control of sexual maturation is a common feature in animal development. A particularly dramatic example is the metamorphosis of insects, in which pulses of the steroid hormone ecdysone drive the wholesale transformation of the larva into an adult. The mechanisms responsible for this transformation are not well understood. Work in Drosophila indicates that the larval and adult forms are patterned by the same underlying sets of developmental regulators, but it is not understood how the same regulators pattern two distinct forms. Recent studies indicate that this ability is facilitated by a global change in the responsiveness of target genes during metamorphosis. This study shows that this shift is controlled in part by the ecdysone-induced transcription factor E93. Although long considered a dedicated regulator of larval cell death, this study found that E93 is expressed widely in adult cells at the pupal stage and is required for many patterning processes at this time. To understand the role of E93 in adult patterning, focus was placed on a simple E93-dependent process, the induction of the Dll gene within bract cells of the pupal leg by EGF receptor signaling. In this system, it was shown that E93 functions to cause Dll to become responsive to EGF receptor signaling. E93 is both necessary and sufficient for directing this switch. E93 likely controls the responsiveness of many other target genes because it is required broadly for patterning during metamorphosis. The wide conservation of E93 orthologs suggests that similar mechanisms control life-cycle transitions in other organisms, including vertebrates (Mou, 2012).

This paper identifies the ecdysone response gene E93 as a key determinant of target gene responsiveness during the pupal phase of metamorphosis. E93 is known to be required for the death of larval tissues, such as the midgut and salivary glands early in metamorphosis, and has been considered a dedicated regulator of cell death. However, this study found that E93 is expressed extensively in imaginal (adult) cells during pupal development and is required for the patterning of many adult structures at this time. Focus was placed on a relatively simple E93-dependent process: the induction of bracts (single-cell pigmented outgrowths) by bristles in the pupal leg. In this system, it was shown that the role of E93 is to render the target gene Distal-less (Dll) responsive to epidermal growth factor receptor (EGFR) signaling. E93 likely controls the pupal-specific responses of many other target genes because it is required broadly for patterning at the pupal stage. This work establishes E93 as a temporal identity factor for adult cells in Drosophila. The conservation of steroid and cell-fate regulatory pathways in diverse taxa suggests that steroid hormones may control life-cycle transitions in vertebrates by similar mechanisms. However, mechanisms underlying the control of adult cell fates by ecdysone have been poorly characterized (Mou, 2012).

In adult cells, many genes seem to undergo a change in their response to specific signaling systems or transcription factors during metamorphosis. Such transitions are most clearly illustrated by recent work showing that targets of the Hox protein Ubx change dramatically at the prepupal and pupal stages. Studies of wing venation and heart remodeling provide additional clear examples, and studies of patterning during metamorphosis in the wing, eye, palp, and abdominal cuticle indicate similar shifts. Such global changes in target responsiveness likely explain how the same signaling systems and identity genes used to pattern the larva during embryogenesis are redeployed during metamorphosis to pattern the adult; however, it is not known how such transitions are controlled (Mou, 2012).

This paper identified the ecdysone response gene E93 as a key determinant of target gene responsiveness during the pupal phase of metamorphosis. E93 is known to be required for the death of larval tissues, such as the midgut and salivary glands early in metamorphosis (Lee, 2000), and has been considered a dedicated regulator of cell death. However, this study found that E93 is expressed extensively in imaginal (adult) cells during pupal development and is required for the patterning of many adult structures at this time. This study focused on a relatively simple E93-dependent process: the induction of bracts (single-cell pigmented outgrowths) by bristles in the pupal leg. In this system, it was shown that the role of E93 is to render the target gene Distal-less (Dll) responsive to epidermal growth factor receptor (EGFR) signaling. E93 likely controls the pupal-specific responses of many other target genes because it is required broadly for patterning at the pupal stage. This work establishes E93 as a temporal identity factor for adult cells in Drosophila. The conservation of steroid and cell-fate regulatory pathways in diverse taxa suggests that steroid hormones may control life-cycle transitions in vertebrates by similar mechanisms (Mou, 2012).

This paper shows that the ecdysone-induced transcription factor E93 specifies pupal-specific target gene responsiveness in adult cells. Relatively little is known about how ecdysone signaling controls adult development at the pupal stage. A few reports have addressed the control of adult cuticle synthesis in the pupa by the ecdysone response gene HR38. However, this study addresses how ecdysone signaling controls adult cell fates at this stage. Focus on a simple and well-characterized patterning event in the pupa: the EGFR-dependent induction of bracts by bristles in the leg. E93 was shown to function as a temporal identity determinant in this process, conferring responsiveness to EGFR signaling upon the Dll gene, which specifies bract fate. The specification of temporal identity by E93 parallels the control of spatial identity by Hox and other selector genes; for some targets, at least, both types of determinant confer competence to respond to specific signaling pathways. Although temporal identity genes have been known for many years in Caenorhabditis elegans, the mode of action suggested in this study (large-scale alteration of target gene response) has not previously been considered (Mou, 2012).

The temporal identity function of E93 is clearly executed by one or both of the known E93 protein isoforms because ectopic expression of either isoform rescues bract formation in E93 mutants, and alleles that truncate these isoforms are defective in this function. How the larval cell-death function of E93 is executed is much less clear. Cell clones homozygous for the E93^1-3 alleles all show normal cuticular patterning, indicating that all specifically affect larval cell death. It has been difficult to understand why the E93^4-6 alleles (nonsense changes at codons 360, 545, and 783, respectively) almost fully complement E93¹ (a nonsense change at codon 995). One possibility is that the cell-death function of E93 is executed by a currently unknown product encoded in part by the region containing the E93¹ change. Resolution of this issue will likely require identification of the sequence changes in the E93² and E93³ alleles (Mou, 2012).

The E93 protein contains a helix-turn-helix DNA binding domain of the Pipsqueak family. This 54-aa domain is highly conserved within the E93 family of orthologs and, for the honey bee ortholog (Mblk-1), has been shown to bind DNA. With one exception, all E93 orthologs contain nuclear receptor interaction motifs (LXXLL motifs). E93 contains three such motifs, suggesting that it binds target enhancers in concert with the EcR or other nuclear receptors induced by ecdysone signaling. In addition, all members of the E93 family contain an interaction motif (PXDLS/TXK/R) for the corepressor C-terminal binding protein (CtBP). The best studied of the E93 orthologs is mammalian ligand-dependent corepressor (LCoR). LCoR was identified as a protein that interacts with hormone-bound estrogen receptor α (White, 2004) but was subsequently shown to interact with a range of ligand-bound nuclear receptors. In most cases, LCoR acts as a corepressor, recruiting both histone deacetylases and CtBP. However, at some targets it acts with CtBP to promote transcription. Structural similarities suggest that E93 will prove to function similarly (Mou, 2012).

The biological roles of the E93 family have not been well-characterized. The C. elegans ortholog (MBR-1) is required for the pruning of excess neurites during the larval stages, and Mblk-1 is expressed within the mushroom bodies, regions of the brain thought to be involved in learning, memory, and sensory integration. In humans, the roles of LCoR are not yet known, but its paralog LCoR-Like has been associated with control of height. Interestingly, two of the insect orthologs of E93 identified are from hemimetabolous species, in which there is no pupal stage. Study of the expression and function of these orthologs promises to shed light on the evolutionary origins of insect metamorphosis (Mou, 2012).

In this analysis of bract induction, it was shown that E93 plays a key role in integrating temporal (ecdysone) and spatial (EGFR) signals. This integration occurs at two levels. First, E93 itself receives inputs from both signals in its expression in bract cells. Ecdysone signaling is required for all imaginal expression of E93 during metamorphosis, whereas EGFR signaling is required only for up-regulation of E93 in bract cells. Second, integration occurs at the level of the Dll gene. Here EGFR signaling plays an instructive role in specifying which epidermal cells express Dll, whereas E93 plays a permissive role, causing Dll to become responsive to EGFR signaling. The requirement for both signals ensures that Dll is activated at only the right place and time (Mou, 2012).

There are two likely ways that Dll could integrate inputs from EGFR signaling and E93. First, E93 and Pnt could both bind to the Dll bract enhancer and cooperate to activate transcription. A second possibility is suggested by the finding that activity of Mblk-1 is modulated by phosphorylation by MAPK, a component of the EGFR signaling pathway. If E93 were similarly modified, signal integration could be achieved simply by the direct activation of Dll by phosphorylated E93. Although this possibility cannot be ruled out, it is thought unlikely for three reasons: First, unlike Mblk-1, E93 has no MAPK consensus phosphorylation sites; second, this mechanism provides no obvious role for Pnt, which is required for Dll activation; and third, E93 is not absolutely essential for activation of Dll. Resolution of which mechanism is used will require identification of an enhancer that requires both E93 and EGFR signaling for its activation (Mou, 2012).

Several of the defects present in E93 mutants result from failures in processes that are regulated by EGFR signaling. In addition to bract induction, these processes include the patterning of wing veins and the pigment cells of the eye. A key step in wing vein development is the activation of the dpp gene in the wing vein primordia during metamorphosis by EGFR signaling. This study found that this activation largely fails in E934 mutants, consistent with a role for E93 in rendering dpp competent to respond to EGFR signaling at the pupal stage. Although these observations suggest a particularly close relationship between E93 and EGFR signaling, other defects in E93 mutants, such as the loss of chemosensory sensilla and patterning abnormalities in the abdominal cuticle, are not clearly related to EGFR signaling and likely result from a failure of target genes to respond to other signaling pathways or transcription factors during metamorphosis (Mou, 2012).

Almost certainly, E93 acts in concert with other factors to confer metamorphosis-specific competence to target genes. The residual expression of Dll in bract cells seen in E93 mutants implies the existence of such additional factors. Moreover, E93 is not expressed in the first 12 h of metamorphosis (the prepupal period), so shifts in target specificity occurring at this stage must be directed by other factors. Several ecdysone response genes are active at this time. This work establishes that E93 is both necessary and sufficient to render the Dll gene responsive to EGFR signaling during metamorphosis. Shifts in target gene specificity directed by E93 likely account in part for how the same selector genes and signaling systems used during embryonic patterning are redeployed during metamorphosis to pattern the adult. Similar mechanisms may operate in humans during the hormonally directed changes of puberty. In addition, shifts in the target specificity of EGFR signaling directed by steroid hormones may play an important role in cancers that depend on both signals, such as many cancers of the breast (Mou, 2012).

Combinatorial activation and repression by seven transcription factors specify Drosophila odorant receptor expression

The mechanism that specifies olfactory sensory neurons to express only one odorant receptor (OR) from a large repertoire is critical for odor discrimination but poorly understood. This study describes the first comprehensive analysis of OR expression regulation in Drosophila. A systematic, RNAi-mediated knock down of most of the predicted transcription factors identified an essential function of acj6, E93, Fer1, onecut, sim, xbp1, and zf30c in the regulation of more than 30 ORs. These regulatory factors are differentially expressed in antennal sensory neuron classes and specifically required for the adult expression of ORs. A systematic analysis reveals not only that combinations of these seven factors are necessary for receptor gene expression but also a prominent role for transcriptional repression in preventing ectopic receptor expression. Such regulation is supported by bioinformatics and OR promoter analyses, which uncovered a common promoter structure with distal repressive and proximal activating regions. Thus, these data provide insight into how combinatorial activation and repression can allow a small number of transcription factors to specify a large repertoire of neuron classes in the olfactory system (Jafari, 2012).

How many OR selector genes are required to uniquely express one OR in each OSN class? Seven OR selector genes were identified, but given the limitations of RNAi, it is likely that there are a total of at least ten critical TFs to specify all OSN classes. Even this probably low estimate generates a rather high number of TFs considering that Drosophila antennae have 34 OSN classes that express ORs. Theoretically the number of TFs needed for a binary combinatorial code to generate 34 unique outcomes is six (2⁶ = 64). Seven TFs can in theory separate 2⁷ = 128 combinations, and ten TFs designate more than 1,000 combinations, suggesting a large number of unused combinations. This surplus of combinations may be due to the inherent randomness of evolution and the impossibility of creating a streamlined code by chance. Another possibility for this large number is the need for a high degree of fidelity, with little or no ectopic OR expression tolerable for proper functioning of the olfactory system. Extrapolation of these observations to the regulatory requirements of the mammalian olfactory system indicates that at least 200-300 TFs would be required to provide a regulatory system that controls >1,000 mammalian ORs, a daunting number. Therefore, it is reasonable to suspect that the stochastic OR selection mechanism found in vertebrates was added during evolution to accommodate the heavy increase in regulatory costs resulting from an expanded number of OR genes (Jafari, 2012).

To date very few TFs have been found to be restricted to small neuronal populations in neuroepithelia or in the developing brain in general. This situation has motivated the suggestion that combinatorial TF regulation defines broad expression patterns of molecules such as neurotransmitters, but is insufficient to generate the large number of neuron classes in, for example, the olfactory system. Similarly, all seven selector genes in this study are expressed across the antenna but still are required for the expression of some few ORs. How can widely expressed TFs then produce restricted expression patterns? Two explanations have been formulated. First, promoter analysis suggests that the OSN class specificity is in part due to repression. Most ORs have a proximal regulatory region next to the gene that is sufficient for expression in OSNs but requires repression from more distal regions for the spatial restriction to each OSN class. In this model, the expression of the TFs that produce OR expression does not need to be particularly specific as long as they are counteracted by repressive factors. Second, the identified TFs can both activate and repress OR expression dependent on the location of the binding site or by the available cofactors. Dual use of the TFs might increase their regulatory power and as a likely consequence the number of TFs required for OR expression to be reduced. It is therefore suggested that specification of large numbers of neuron classes in the olfactory system and likely in the nervous system, require two layers of combinatorial coding, one layer of terminal selector genes that produce expression and a layer of repressors that restrict the expression to each class (Jafari, 2012).

EVOLUTIONARY HOMOLOGS

Mushroom bodies (MBs) are considered to be involved in higher-order sensory processing in the insect brain. To identify the genes involved in the intrinsic function of the honeybee MBs, genes preferentially expressed therein were sought using the differential display method. A novel gene encoding a putative transcription factor (Mblk-1) is expressed preferentially in one of two types of intrinsic MB neurons -- the large-type Kenyon cells. Mblk-1 is thus a candidate gene involved in the advanced behaviours of honeybees. A putative DNA binding motif of Mblk-1 has significant sequence homology with those encoded by genes from various animal species, suggesting that the functions of these proteins in neural cells are conserved among the animal kingdom (Takeuchi, 2001).

The Mblk-1 gene in the honeybee brain encodes a transcription factor containing two DNA binding motifs, termed RHF1 and 2. Two mouse Mblk1 homologs, Mlr1 and Mlr2, have been identified. Both encode proteins containing a single DNA-binding motif highly conserved with RHF2, and both activate transcription mediated by a DNA element recognized by honeybee Mblk-1. Mlr1 is expressed predominantly in the spermatocytes of the testis, while Mlr2 is expressed in various tissues other than testis. Mlr1 transcripts are lost in the testis of W/W(v) mutant mice, suggesting a role in spermatogenesis (Kunieda, 2003).

Knockdown of Ecdysone-Induced Protein 93F Causes Abnormal Pupae and Adults in the Eggplant Lady Beetle

Ecdysone-induced protein 93F (E93) plays triple roles during post-embryonic development in insects whose juvenile instars are more than four. However, it only acts as a specifier of adult structures in Drosophila flies whose larval instars are fixed at three. In this study, the functions were determined of E93 in the eggplant lady beetle (Henosepilachna vigintioctopunctata), which has four larval instars. E93 was shown to be abundantly expressed at the prepupal and pupal stages. A precocious inhibition of the juvenile hormone signal by RNA interference (RNAi) of HvKr-h1 or HvHairy, two vital downstream developmental effectors, at the penultimate instar larval stage increased the expression of E93, Conversely, ingestion of JH by the third-instar larvae stimulated the expression of HvKr-h1 but repressed the transcription of either HvE93X1 or HvE93X2. However, disturbance of the JH signal neither drove premature metamorphosis nor caused supernumerary instars. In contrast, depletion of E93 at the third- and fourth-instar larval and prepupal stages severely impaired pupation and caused a larval-pupal mixed phenotype: pupal spines and larval scoli were simultaneously presented on the cuticle. RNAi of E93 at the pupal stage affected adult eclosion. When the beetles had suffered from a dsE93 injection at the fourth-instar larval and pupal stages, a few resultant adults emerged, with separated elytra, abnormally folded hindwings, a small body size and short appendages. Taken together, these results suggest the larval instars are fixed in H. vigintioctopunctata; E93 serves as a repressor of larval characters and a specifier of adult structures during the larval-pupal-adult transition.

REFERENCES

Search PubMed for articles about Drosophila Ecdysone-induced protein 93F

Baehrecke, E. H. and Thummel, C. S. (1995). The Drosophila E93 gene from the 93F early puff displays stage- and tissue-specific regulation by 20-hydroxyecdysone. Dev. Biol. 171: 85-97. 7556910

Broadus, J., et al. (1999). The Drosophila beta FTZ-F1 orphan nuclear receptor provides competence for stage-specific responses to the steroid hormone ecdysone. Mol. Cell 3(2): 143-9. PubMed Citation: 10078197

Chronis, C., Fiziev, P., Papp, B., Butz, S., Bonora, G., Sabri, S., Ernst, J. and Plath, K. (2017). Cooperative binding of transcription factors orchestrates reprogramming. Cell 168(3): 442-459 e420. PubMed ID: 28111071

Daish, T. J., Cakouros, D. and Kumar, S. (2003). Distinct promoter regions regulate spatial and temporal expression of the Drosophila caspase dronc. Cell Death Dif. 10: 1348-1356. 12970673

Gorski, S. M., et al. (2003). A SAGE approach to discovery of genes involved in autophagic cell death. Curr. Biol. 13(4): 358-63. 12593804

Jafari, S., et al. (2012). Combinatorial activation and repression by seven transcription factors specify Drosophila odorant receptor expression. PLoS Biol. 10(3): e1001280. PubMed Citation: 22427741

Jiang, C., Baehrecke, E. H. and Thummel, C. S. (1997). Steroid regulated programmed cell death during Drosophila metamorphosis. Development 124: 4673-4683. 9409683

Kunieda, T., Park, J. M., Takeuchi, H. and Kubo, T. (2003). Identification and characterization of Mlr1,2: two mouse homologues of Mblk-1, a transcription factor from the honeybee brain. FEBS Lett. 535(1-3): 61-65. 12560079

Lee, C.-Y., et al. (2000). E93 directs steroid-triggered programmed cell death in Drosophila. Mol. Cell 6: 433-443. 10983989

Lee, C.-Y. and Baehrecke, E. H. (2001). Steroid regulation of autophagic programmed cell death during development. Development 128: 1443-1455. 11262243

Lee, C.-Y., Cooksey, B. A. and Baehrecke, E. H. (2002). Steroid regulation of midgut cell death during Drosophila development. Dev. Bio. 250: 101-111. 12297099

Lee, C. Y., et al. (2003). Genome-wide analyses of steroid- and radiation-triggered programmed cell death in Drosophila. Curr. Biol. 13: 350-357. 12593803

Mou, X., Duncan, D. M., Baehrecke, E. H. and Duncan, I. (2012). Control of target gene specificity during metamorphosis by the steroid response gene E93. Proc. Natl. Acad. Sci. 109(8): 2949-54. PubMed Citation: 22308414

Martin, D. N. and Baehrecke, E. H. (2004). Caspases function in autophagic programmed cell death in Drosophila. Development 131: 275-284. 14668412

Richards G., et al. (1999). The acquisition of competence to respond to ecdysone in Drosophila is transcript specific. Mech. Dev. 82(1-2): 131-139. PubMed Citation: 10354477

Siegmund, T. and Lehmann, M. (2002). The Drosophila Pipsqueak protein defines a new family of helix-turn-helix DNA-binding proteins. Dev. Genes Evol. 212(3): 152-7. 11976954

Syed, M. H., Mark, B. and Doe, C. Q. (2017). Steroid hormone induction of temporal gene expression in Drosophila brain neuroblasts generates neuronal and glial diversity. Elife 6 [Epub ahead of print]. PubMed ID: 28394252

Takeuchi, H., et al. (2001). Identification of a novel gene, Mblk-1, that encodes a putative transcription factor expressed preferentially in the large-type Kenyon cells of the honeybee brain. Insect Mol. Biol. 10(5): 487-94. 11881813

Uyehara, C. M., Nystrom, S. L., Niederhuber, M. J., Leatham-Jensen, M., Ma, Y., Buttitta, L. A. and McKay, D. J. (2017). Hormone-dependent control of developmental timing through regulation of chromatin accessibility. Genes Dev 31(9):862-875. PubMed ID: 28536147

White, J. H., Fernandes, I., Mader, S. and Yang, X-J. (2004). Corepressor recruitment by agonist-bound nuclear receptors. Vitam. Horm. 68: 123-143. PubMed Citation: 15193453

Woodard, C. T., Baehrecke, E. H. and Thummel, C. S. (1994). A molecular mechanism for the stage specificity of the Drosophila prepupal genetic response to ecdysone. Cell 79: 607-615. 7954827

Wu, J. J., Chen, F., Yang, R., Shen, C. H., Ze, L. J., Jin, L. and Li, G. Q. (2022). Knockdown of Ecdysone-Induced Protein 93F Causes Abnormal Pupae and Adults in the Eggplant Lady Beetle. Biology (Basel) 11(11). PubMed ID: 36358341

Yokoyama, H., Mukae, N., Sakahira, H., Okawa, K., Iwamatsu, A., and Nagata, S. (2000). A novel activation mechanism of caspaseactivated DNase from Drosophila melanogaster. J. Biol. Chem. 275: 12978-12986. 10777599

date revised: 22 August 2023

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