Ecdysone-induced protein 74EF

DEVELOPMENTAL BIOLOGY

Embryonic

E74A is transcribed at the end of embryogenesis at a stage that is not associated with ecdysone pulses. It is also transcribed in an ecdysone independent fashion, at the end of pupal development, a period that extends to the adult stage (Thummel, 1990).

Larval

The E74 gene is responsible for the early ecdysone-inducible puff at position 74EF and encodes two related DNA-binding proteins that appear to play a regulatory role in the hierarchy. E74A is expressed in a wide variety of late-third instar tissues, suggesting that it plays a broad pleiotropic role in response to the hormone. In larval tissues E74A mRNA is concentrated over the large polytene nuclei. This nuclear localization is most obvious in the salivary gland, although it can also be detected in the polytene nuclei of fat bodies, intestine, proventriculus, epidermis and muscle. During early prepupae, when the overall levels of E74A mRNA are decreasing, relatively high levels of E74AmRNA persist in the gut, peripodial membranes of the imaginal discs, and proliferation centers of the brain. E74A mRNA is present in eye-antennal, wing and leg imaginal discs as well as genital and haltere discs. In the larval brain, E74A mRNA is preferentially expressed in specific cell types, including the glial cells surrounding the neuropil and the proliferation centers. Expression of E74A mRNA is detected in mouth part muscles, ring gland , trachea, Malpighian tubules, proventriculus, gastric caecae and mid-intestine. E74A mRNA in the proliferation centers of the brain does not appear to be translated. The spatial distribution of nuclear E74A protein correlates with its the RNA distribution, with the single exception that no E74A protein can be detected in the proliferation centers of the brain. There is also a temporal discrepancy between E74A mRNA and protein accumulation. The peak of E74A protein induced by the late larval ecdysone pulse follows the peak of E74A mRNA by approximately 2 h. This delay is not seen at 10 h prepupae, when the next pulse of ecdysone induces the simultaneous expression of E74A mRNA and protein. The unusually long and complex 5' leader in the E74A mRNA may regulate its translation (Boyd, 1991).

Effects of mutation or deletion

During Drosophila third instar larval development, one or more pulses of the steroid hormone ecdysone activate three temporally distinct sets of genes in the salivary glands, represented by puffs in the polytene chromosomes. The intermolt genes are induced first, in mid-third instar larvae: these genes encode a protein glue used by the animal to adhere itself to a solid substrate for metamorphosis. The intermolt genes are repressed at puparium formation as a high titer ecdysone pulse directly induces a small set of early regulatory genes. The early genes both repress their own expression and activate more than 100 late secondary-response genes. broad (Broad-Complex) is an early ecdysone-inducible gene that encodes a family of DNA binding proteins defined by at least three lethal complementation groups: br, rbp, and l(1)2Bc. BRC is critical for the appropriate regulation of all three classes of ecdysone-inducible genes. Both rbp and l(1)2Bc are required for glue gene induction in mid-third instar larvae. In addition, the l(1)2Bc function is required for glue gene repression in prepupae; in l(1)2Bc mutants the glue genes are re-induced by the late prepupal ecdysone pulse, recapitulating a mid-third instar regulatory response at an inappropriate stage in development. l(1)2Bc function is also required for the complete ecdysone induction of some early mRNAs (E74A, E75A, and BRC) and efficient repression of most early mRNAs in prepupae. Like the intermolt secondary-response genes, the late secondary-response genes are absolutely dependent on rbp for their induction. An effect of l(1)2Bc mutations on late gene activity can also be detected, but is most likely a secondary consequence of the submaximal ecdysone-induction of a subset of early regulatory products. These results indicate that BRC plays a key role in dictating the stage-specificity of the ecdysone response. In addition, the Ecdysone-receptor protein complex alone is not sufficient for appropriate induction of the early primary-response genes, but requires the prior expression of BR-C proteins. These studies define the BR-C as a key regulator of gene activity at the onset of metamorphosis in Drosophila (Karim, 1993).

broad and E74 are induced directly by ecdysone and encode families of transcription factors that regulate ecdysone primary- and secondary-response genes. Genetic analyses have revealed that mutations in BRC and E74 are lethal during metamorphosis; these mutations also cause some similar lethal phenotypes and alterations in secondary-response gene transcription. To examine whether BRC and E74 function together during development, representative alleles from each BRC and E74 complementation group have been combined. Analysis of the morphological and molecular phenotypes of the double-mutant animals reveals that BRC and E74 alleles do act together, producing both novel and synergistic effects. BRC and E74 share functions in puparium formation, pupation and early gene induction. In addition, the BR-C and E74 transcription factors may directly interact to regulate the expression of salivary gland glue and late genes (Fletcher, 1995a).

Steroid regulation of autophagic programmed cell death during development

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 (Apaf-1-related-killer), 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).

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date revised:  10 December 2014

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