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).
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)93FX2, 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)93FX2 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).
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).
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date revised: 10 July 2005
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